Bi-functional chimeric proteins and uses thereof

ABSTRACT

The present invention relates, in part, to targeted chimeric proteins with beneficial therapeutic effects, including, for example, effects mediated by mutant forms of soluble agents that are part of the chimeric proteins. Pharmaceutical compositions comprising the chimeric proteins are also provided. The present invention finds use in the treatment of various disease and disorders.

RELATED APPLICATIONS

This application is a U.S. National Stage (371) entry of anInternational Patent Application PCT/IB2016/001668, filed Nov. 7, 2016,which claims the benefit of and priority to U.S. Provisional PatentApplication No. 62/252,282, filed Nov. 6, 2015, U.S. Provisional PatentApplication No. 62/252,286, filed Nov. 6, 2015, and U.S. ProvisionalPatent Application No. 62/335,868, filed May 13, 2016, the entiredisclosures of all of which are hereby incorporated herein by referencein their entireties.

FIELD

The present invention relates, in part, to targeted chimeric proteinswith beneficial therapeutic properties. The present invention alsoprovides use of the chimeric proteins in pharmaceutical compositions andmethods of treating various diseases and disorders.

DESCRIPTION OF THE TEXT FILE SUBMITTED ELECTRONICALLY

The contents of the text file submitted electronically herewith areincorporated herein by reference in their entirety: a computer readableformat copy of the Sequence Listing (filename:ORN-007PC_Sequence_listing.txt; date recorded: Nov. 3, 2016; file size:300 KB).

BACKGROUND

Soluble agents such as cytokines, hormones, and growth factors arenaturally occurring substances capable of modulating cellular growth anddifferentiation. These soluble agents play important roles in a varietyof physiological processes including, for example, metabolism,respiration, sleep, excretion, healing, movement, reproduction, mood,stress, tissue function, immune function, sensory perception, and growthand development. Typically, cytokines, hormones, and growth factorsexert their biological effects through binding to specific receptors onthe surface of target cells.

Clinically, cytokines, hormones, and growth factors are used in thetreatment of a variety of diseases and disorders including, for example,cancers, microbial infections, hematological disorders, and metabolicdiseases. Despite this common use, the administration of these solubleagents is not without risks. The therapeutic use of cytokines, hormones,and growth factors is often associated with systemic toxicity anddeleterious side effects thus limiting the dose levels that these agentscan be used. As a result, only relatively small numbers of cytokines arecurrently approved by regulatory agencies. Of these, fourteen of theFDA-approved cytokine preparations carry warnings, ten of which areblack box warnings.

Furthermore, and relatedly, many of these soluble agents havepromiscuous binding activity and therefore provide for the possibilityof off-target effects, which can underlie deleterious side effects or,at the least, provide a sink for the therapeutic construct away from thesite of therapeutic action.

There is a need to develop therapeutic soluble agents with improvedsafety and efficacy.

SUMMARY

Accordingly, the present invention provides, in various aspects,chimeric proteins that include a soluble agent which has reducedaffinity at a therapeutic receptor, which allows for attenuation ofbinding and/or activity (inclusive of agonism or antagonism), andsubstantially reduced or ablated affinity at a second receptor, which,for example, prevents or substantially reduces non-therapeutic signalingor undesirable sequestration of the chimeric protein. In the case of theattenuated activity at the therapeutic receptor, the weakened affinityat the therapeutic receptor is restorable by attachment to a targetingmoiety (e.g. an antibody or antibody format described herein), suchtargeting moiety having high affinity for an antigen at the site oftherapeutic activity. In this way, the present chimeric proteinsprovide, in some embodiments, localized, on-target, and controlledtherapeutic action at the therapeutic receptor and avoid activity atanother receptor that could diminish or interfere with the overalltherapeutic effect. Accordingly, in various aspects, the presentchimeric constructs make for more therapeutically effective solubleagents by, for example, reducing receptor promiscuity and attenuatingactivity at the desired therapeutic receptor.

In various embodiments, the soluble agent is modified to have a mutationthat reduces its binding affinity or activity at a therapeutic receptor.This binding affinity or activity is restorable by connection with atargeting moiety, e.g. an antibody or antibody format described herein,and binding of the targeting moiety to an antigen or epitope at a siteof therapeutic activity (e.g. a cell as described herein). In someembodiments, the activity provided by the soluble agent is agonism atthe therapeutic receptor (e.g. activation of a cellular effect at a siteof therapy). In some embodiments, the activity provided by the solubleagent is antagonism at the therapeutic receptor (e.g. blocking ordampening of a cellular effect at a site of therapy).

In various embodiments, the therapeutic chimeric proteins of the presentinvention reduce off-target effects because their soluble agents havemutations that weaken binding affinity or activity at a therapeuticreceptor. In various embodiments, this reduces side effects observedwith, for example, wild type soluble agents. However, in variousembodiments, the present chimeric proteins also have soluble agents withmutations that substantially reduce or ablate binding or activity atanother receptor. This, in some embodiments, further reduces off-targeteffects of the present chimeric proteins and therefore reduces sideeffects (e.g. relative to wild type soluble agents). In variousembodiments, this affinity or activation at the other receptor is notrestorable with a targeting moiety. In various embodiments,substantially reducing or ablating binding or activity at anotherreceptor also prevents deleterious effects that are mediated by anotherreceptor. Alternatively, or in addition, substantially reducing orablating binding or activity at another receptor causes the therapeuticeffect to improve as there is a reduced or eliminated sequestration ofthe therapeutic chimeric proteins away from the site of therapeuticaction and, optionally, the ability to reduce dose of the therapeuticchimeric proteins. In various embodiments, the dual effect at atherapeutic receptor and another receptor can be mediated by the samemutation or different mutations, in various number and varieties asdescribed herein.

In various embodiments, the present chimeric proteins find use in thetreatment of various diseases or disorders and the present inventionencompasses various methods of treatment.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 shows, in panels A and B, results from cytotoxicity assays usingMCF cells. Specifically, the cytotoxicity of various human TNF-R1selective mutants was tested in MCF cells (panel A) or hCD20-expressingMCF cells (panel B). FIG. 1 , panel C, shows results from assays usingRaji cells, in which the ability of the various human TNF-R1 selectivemutants to induce OPRM1 expression was assessed.

FIG. 2 shows, in panels A and B, results from cytotoxicity assays usingMCF cells. Specifically, the cytotoxicity of various human TNF-R1selective mutants was tested. FIG. 2 , panel C, shows results fromassays using Raji cells, in which the ability of the various humanTNF-R1 selective mutants to induce OPRM1 expression was assessed.

FIG. 3 shows, in panels A and B, results from cytotoxicity assays usingMCF cells. Specifically, the cytotoxicity of various human TNF-R2selective mutants was tested. FIG. 3 , panel C, shows results fromassays using Raji cells, in which the ability of the various humanTNF-R2 selective mutants to induce OPRM1 expression was assessed.

FIG. 4 , panel A, shows results from cytotoxicity assays using MCFcells. Specifically the cytotoxicity of various hTNF mutants was tested.FIG. 4 , panel B, shows results from GM-CSF secretion assays usingPC60-hTNFR2 cells.

DETAILED DESCRIPTION

The present invention is based, in part, on the surprising discoverythat specifically targeted chimeric proteins that include a modifiedsoluble agent with reduced affinity for one or more receptors exhibitbeneficial therapeutic properties and reduced side effects. In variousembodiments, these targeted chimeric proteins provide controlledactivity at a therapeutic receptor and reduced promiscuity of receptorinteractions (e.g. by substantially reducing or ablating binding atanother receptor). The present invention provides pharmaceuticalcompositions comprising the chimeric proteins and their use in thetreatment of various diseases. Administration of the chimeric proteinsand pharmaceutical compositions of the invention achieves significantlyreduced side effects compared to the wild type soluble agent.

Modified Soluble Agent

In one aspect, the present invention provides a chimeric protein thatincludes a soluble agent which has reduced affinity at a therapeuticreceptor, which allows for attenuation of activity (inclusive of agonismor antagonism), and substantially reduced or ablated affinity at asecond receptor, which, for example, prevents non-therapeutic signalingor undesirable sequestration of the chimeric protein.

In various embodiments, the attenuated activity at the therapeuticreceptor, the weakened affinity at the therapeutic receptor isrestorable by attachment with a targeting moiety, having high affinityfor an antigen at the site of therapeutic activity (e.g. an antibody orantibody format described herein). The targeting is realized by linkingthe modified soluble agent to a targeting moiety. In an embodiment, themodified soluble agent is linked to a targeting moiety through itsamino-terminus. In another embodiment, the modified soluble agent islinked to a targeting moiety through its carboxy-terminus. In this way,the present chimeric proteins provide, in some embodiments, localized,on-target, and controlled therapeutic action at the therapeutic receptorand avoid activity at a second receptor that could diminish or interferewith the overall therapeutic effect. Accordingly, in various aspects,the present chimeric constructs make for more therapeutically effectivesoluble agents by reducing receptor promiscuity and attenuating activityat the desired therapeutic receptor.

In various embodiments, the soluble agent is selected from modifiedversions of cytokines, growth factors, and hormones. Illustrativeexamples of such cytokines, growth factors, and hormones include, butare not limited to, lymphokines, monokines, traditional polypeptidehormones, such as human growth hormone, N-methionyl human growthhormone, and bovine growth hormone; parathyroid hormone; thyroxine;insulin; proinsulin; relaxin; prorelaxin; glycoprotein hormones such asfollicle stimulating hormone (FSH), thyroid stimulating hormone (TSH),and luteinizing hormone (LH); hepatic growth factor; fibroblast growthfactor; prolactin; placental lactogen; tumor necrosis factor-α and tumornecrosis factor-β; mullerian-inhibiting substance; mousegonadotropin-associated peptide; inhibin; activin; vascular endothelialgrowth factor; integrin; thrombopoietin (TPO); nerve growth factors suchas NGF-α; platelet-growth factor; transforming growth factors (TGFs)such as TGF-α and TGF-β; insulin-like growth factor-I and -II; osteoinductive factors; interferons such as, for example, interferon-α,interferon-β and interferon-γ (and interferon type I, II, and III),colony stimulating factors (CSFs) such as macrophage-CSF (M-CSF);granulocyte-macrophage-CSF (GM-CSF); and granulocyte-CSF (G-CSF);interleukins (ILs) such as, for example, IL-1, IL-1α, IL-2, IL-3, IL-4,IL-5, IL-6, IL-7, IL-8, IL-9, IL-10, IL-11, IL-12, IL-13, and IL-18; atumor necrosis factor such as, for example, TNF-α or TNF-β; and otherpolypeptide factors including, for example, LIF and kit ligand (KL). Asused herein, cytokines, growth factors, and hormones include proteinsobtained from natural sources or produced from recombinant bacterial,eukaryotic or mammalian cell culture systems and biologically activeequivalents of the native sequence cytokines.

In some embodiments, the soluble agent is a modified version of a growthfactor selected from, but not limited to, transforming growth factors(TGFs) such as TGF-α and TGF-β, epidermal growth factor (EGF),insulin-like growth factor such as insulin-like growth factor-I and -II,fibroblast growth factor (FGF), heregulin, platelet-derived growthfactor (PDGF), vascular endothelial growth factor (VEGF).

In an embodiment, the growth factor is a modified version of afibroblast growth factor (FGF). Illustrative FGFs include, but are notlimited to, FGF1, FGF2, FGF3, FGF4, FGF5, FGF6, FGF7, FGF8, FGF9, FGF10,FGF11, FGF12, FGF13, FGF14, murine FGF15, FGF16, FGF17, FGF18, FGF19,FGF20, FGF21, FGF22, and FGF23.

In an embodiment, the growth factor is a modified version of a vascularendothelial growth factor (VEGF). Illustrative VEGFs include, but arenot limited to, VEGF-A, VEGF-B, VEGF-C, VEGF-D, and PGF and isoformsthereof including the various isoforms of VEGF-A such as VEGF₁₂₁,VEGF₁₂₁b, VEGF₁₄₅, VEGF₁₆₅, VEGF₁₆₅b, VEGF₁₈₉, and VEGF₂₀₆.

In an embodiment, the growth factor is a modified version of atransforming growth factor (TGF). Illustrative TGFs include, but are notlimited to, TGF-α and TGF-β and subtypes thereof including the varioussubtypes of TGF-β including TGFβ1, TGFβ2, and TGβ3.

In some embodiments, the soluble agent is a modified version of ahormone selected from, but not limited to, human chorionic gonadotropin,gonadotropin releasing hormone, an androgen, an estrogen,thyroid-stimulating hormone, follicle-stimulating hormone, luteinizinghormone, prolactin, growth hormone, adrenocorticotropic hormone,antidiuretic hormone, oxytocin, thyrotropin-releasing hormone, growthhormone releasing hormone, corticotropin-releasing hormone,somatostatin, dopamine, melatonin, thyroxine, calcitonin, parathyroidhormone, glucocorticoids, mineralocorticoids, adrenaline, noradrenaline,progesterone, insulin, glucagon, amylin, calcitriol, calciferol,atrial-natriuretic peptide, gastrin, secretin, cholecystokinin,neuropeptide Y, ghrelin, PYY3-36, insulin-like growth factor (IGF),leptin, thrombopoietin, erythropoietin (EPO), and angiotensinogen.

In some embodiments, the soluble agent is a modified version of aninterleukin selected from, but not limited to, IL-I, IL-2, IL-3, IL-4,IL-5, IL-6, IL-7, IL-8, IL-9, IL-10, IL-11, IL-12, IL-13, IL-14, IL-15,IL-16, IL-17, IL-18, IL-19, IL-20, IL-21, IL-22, IL-23, IL-24, IL-25,IL-26, IL-27, IL-28, IL-29, IL-30, IL-31, IL-32, IL-33, IL-34, IL-35,and/or, IL-36.

In some embodiments, the soluble agent is a modified version of aninterferon such as interferon types I, II, and III. Illustrativeinterferons, including for example, interferon-α-1, 2, 4, 5, 6, 7, 8,10, 13, 14, 16, 17, and 21, interferon-β and interferon-γ, interferon κ,interferon ε, interferon τ, and interferon ω.

In some embodiments, the soluble agent is a modified version of a tumornecrosis factor (TNF) or a protein in the TNF family, including but notlimited to, TNF-α, TNF-β, CD40L, CD27L, CD30L, FASL, 4-1BBL, OX40L, andTRAIL.

In various embodiments, the soluble agent is modified to have one ormore mutations. In some embodiments, the mutations allow for themodified soluble agent to have one or more of attenuated activity suchas one or more of reduced binding affinity, reduced endogenous activity,and reduced specific bioactivity relative to unmutated, e.g., the wildtype form of the soluble agent. For instance, one or more of attenuatedactivity such as reduced binding affinity, reduced endogenous activity,and reduced specific bioactivity relative to unmutated, e.g. the wildtype form of the soluble agent may be at a therapeutic receptor and asecond receptor. Consequentially, in various embodiments, the mutationsallow for the modified soluble agent to have reduced systemic toxicity,reduced side effects, and reduced off-target effects relative tounmutated, e.g. the wild type form of the soluble agent.

In various embodiments, the soluble agent is modified to have a mutationthat reduces its binding affinity or activity at a therapeutic receptor.In some embodiments, the activity provided by the wild type solubleagent is agonism at the therapeutic receptor (e.g. activation of acellular effect at a site of therapy). For example, the wild typesoluble agent may activate the therapeutic receptor. In suchembodiments, the mutations result in the modified soluble agent to havereduced activating activity at the therapeutic receptor. In someembodiments, the activity provided by the wild type soluble agent isantagonism at the therapeutic receptor (e.g. blocking or dampening of acellular effect at a site of therapy). For example, the wild typesoluble agent may antagonize or inhibit the therapeutic receptor. Inthese embodiments, the mutations result in the modified soluble agent tohave reduced antagonizing activity at the therapeutic receptor. In someembodiments, the reduced affinity or activity at the therapeuticreceptor is restorable by attachment with a targeting moiety. In otherembodiments, the reduced affinity or activity at the therapeuticreceptor is not substantially restorable by the activity of thetargeting moiety. In various embodiments, the therapeutic chimericproteins of the present invention reduce off-target effects becausetheir soluble agents have mutations that weaken binding affinity oractivity at a therapeutic receptor. In various embodiments, this reducesside effects observed with, for example, the wild type soluble agents.In various embodiments, the modified soluble agent is substantiallyinactive en route to the site of therapeutic activity and has its effectsubstantially on specifically targeted cell types which greatly reducesundesired side effects.

In various embodiments, the present chimeric proteins also have solubleagents with mutations that substantially reduce or ablate binding oractivity at another receptor. This, in some embodiments, further reducesoff-target effects of the present chimeric proteins and thereforereduces side effects. In some embodiments, this substantial reduction orablation of binding or activity is not restorable with a targetingmoiety. In various embodiments, substantially reducing or ablatingbinding or activity at a second receptor also may prevent deleteriouseffects that are mediated by the other receptor. Alternatively, or inaddition, substantially reducing or ablating binding or activity at theother receptor causes the therapeutic effect to improve as there is areduced or eliminated sequestration of the therapeutic chimeric proteinsaway from the site of therapeutic action. For instance, in someembodiments, this obviates the need of high doses of the presentchimeric proteins that compensate for loss of binding or activity at theother receptor. Such ability to reduce dose further provides a lowerlikelihood of side effects.

In some embodiments, the chimeric proteins have modified soluble agentsbearing a mutation that affects interaction with a therapeutic receptorand another receptor (e.g. mediated by the same mutation or multiplemutations). In some embodiments, the present chimeric proteins have amodified soluble agent that has both mutations that attenuate bindingand/or activity at a therapeutic receptor and therefore allow for a morecontrolled, on-target therapeutic effect (e.g. relative wild typesoluble agent) and mutations that substantially reduce or ablate bindingand/or activity at another receptor and therefore reduce side effects(e.g. relative the wild type soluble agent). These mutations may be atthe same or at different positions.

In various embodiments, the dual effect at a therapeutic receptor andanother receptor can be mediated by the same mutation or multiplemutations. In various embodiments, the mutation(s) that reduce bindingand/or activity at a therapeutic receptor is different than themutation(s) that substantially reduce or ablate at another receptor. Invarious embodiments, the mutation(s) that reduce binding and/or activityat a therapeutic receptor are the same as the mutation(s) thatsubstantially reduce or ablate at another receptor.

In various embodiments, the modified soluble agent comprises one or moremutations that cause the soluble agent to have attenuated or reducedaffinity, e.g. binding (e.g. K_(D)) and/or activation (for instance,when the modified soluble agent is an agonist at the therapeuticreceptor, measurable as, for example, K_(A) and/or EC₅₀) and/orinhibition (for instance, when the modified soluble agent is anantagonist at the therapeutic receptor, measurable as, for example,K_(I) and/or IC₅₀), for one or more therapeutic receptors. In variousembodiments, the reduced affinity at the therapeutic receptor allows forattenuation of activity (inclusive of agonism or antagonism). In suchembodiments, the modified soluble agent has about 1%, or about 3%, about5%, about 10%, about 15%, about 20%, about 25%, about 30%, about 35%,about 40%, about 45%, about 50%, about 60%, about 65%, about 70%, about75%, about 80%, about 85%, about 90%, about 95%, or about 10%-20%, about20%-40%, about 50%, about 40%-60%, about 60%-80%, about 80%-100% of theaffinity for the therapeutic receptor relative to the wild type solubleagent. In some embodiments, the binding affinity is at least about2-fold lower, about 3-fold lower, about 4-fold lower, about 5-foldlower, about 6-fold lower, about 7-fold lower, about 8-fold lower, about9-fold lower, at least about 10-fold lower, at least about 15-foldlower, at least about 20-fold lower, at least about 25-fold lower, atleast about 30-fold lower, at least about 35-fold lower, at least about40-fold lower, at least about 45-fold lower, at least about 50-foldlower, at least about 100-fold lower, at least about 150-fold lower, orabout 10-50-fold lower, about 50-100-fold lower, about 100-150-foldlower, about 150-200-fold lower, or more than 200-fold lower relative tothe wild type soluble agent.

In various embodiments, the modified soluble agent comprises one or moremutations that cause the soluble agent to have substantially reduced orablated affinity, e.g. binding (e.g. K_(D)) and/or activation (forinstance, when the modified soluble agent is an agonist at thetherapeutic receptor, measurable as, for example, K_(A) and/or EC₅₀)and/or inhibition (for instance, when the modified soluble agent is anantagonist at the therapeutic receptor, measurable as, for example,K_(I) and/or IC₅₀), for one or more other receptors. In suchembodiments, the modified soluble agent has about 1%, or about 3%, about5%, about 10%, about 15%, about 20%, about 25%, about 30%, about 35%,about 40%, about 45%, about 50%, about 60%, about 65%, about 70%, about75%, about 80%, about 85%, about 90%, about 95%, or about 10%-20%, about20%-40%, about 50%, about 40%-60%, about 60%-80%, about 80%-100% of theaffinity for the other receptor relative to the wild type soluble agent.In some embodiments, the binding affinity is at least about 2-foldlower, about 3-fold lower, about 4-fold lower, about 5-fold lower, about6-fold lower, about 7-fold lower, about 8-fold lower, about 9-foldlower, at least about 10-fold lower, at least about 15-fold lower, atleast about 20-fold lower, at least about 25-fold lower, at least about30-fold lower, at least about 35-fold lower, at least about 40-foldlower, at least about 45-fold lower, at least about 50-fold lower, atleast about 100-fold lower, at least about 150-fold lower, or about10-50-fold lower, about 50-100-fold lower, about 100-150-fold lower,about 150-200-fold lower, or more than 200-fold lower relative to thewild type soluble agent.

In various embodiments, the attenuation or reduction in binding affinityof a modified soluble agent for the therapeutic receptor is less thanthe substantial reduction or ablation in affinity for the otherreceptor. In some embodiments, the attenuation or reduction in bindingaffinity of a modified soluble agent for the therapeutic receptor isless than the substantial reduction or ablation in affinity for theother receptor by about 1%, or about 3%, about 5%, about 10%, about 15%,about 20%, about 25%, about 30%, about 35%, about 40%, about 45%, about50%, about 60%, about 65%, about 70%, about 75%, about 80%, about 85%,about 90%, or about 95%. In various embodiments, substantial reductionor ablation refers to a greater reduction in binding affinity and/oractivity than attenuation or reduction.

In various embodiments, the modified soluble agent comprises one or moremutations that reduce the endogenous activity of the soluble agent toabout 75%, or about 70%, or about 60%, or about 50%, or about 40%, orabout 30%, or about 25%, or about 20%, or about 10%, or about 5%, orabout 3%, or about 1%, e.g., relative to the wild type soluble agent

In various embodiments, the modified soluble agent comprises one or moremutations that cause the soluble agent to have reduced affinity and/oractivity for a receptor of any one of the cytokines, growth factors, andhormones as described herein. In such embodiments, the modified solubleagent comprises one or more mutations that cause the soluble agent tohave substantially reduced or ablated affinity and/or activity for adifferent receptor of any one of the cytokines, growth factors, andhormones as described herein.

In some embodiments, the modified soluble agent comprises one or moremutations that cause the modified soluble agent to have reduced affinityfor a receptor. In some embodiments, the modified soluble agent affinityfor a receptor is lower than the binding affinity of the targetingmoiety for its receptor. In some embodiments, this binding affinitydifferential is between the modified soluble agent/receptor andtargeting moiety/receptor on the same cell. In some embodiments, thisbinding affinity, differential allows for the modified soluble agent tohave localized, on-target effects and to minimize off-target effectsthat underlie side effects that are observed with wild type solubleagents. In some embodiments, this binding affinity is at least about2-fold, or at least about 5-fold, or at least about 10-fold, or at leastabout 15-fold lower, or at least about 25-fold, or at least about50-fold lower, or at least about 100-fold, or at least about 150-foldless.

Receptor binding activity may be measured using methods known in theart. See, for example, Elliott S., et al., (1997) Blood, 89:493-502, theentire contents of which are hereby incorporated by reference.

The amino acid sequences of the wild type soluble agents describedherein are well known in the art. Accordingly, in various embodimentsthe modified soluble agent comprises an amino acid sequence that has atleast about 60%, or at least about 61%, or at least about 62%, or atleast about 63%, or at least about 64%, or at least about 65%, or atleast about 66%, or at least about 67%, or at least about 68%, or atleast about 69%, or at least about 70%, or at least about 71%, or atleast about 72%, or at least about 73%, or at least about 74%, or atleast about 75%, or at least about 76%, or at least about 77%, or atleast about 78%, or at least about 79%, or at least about 80%, or atleast about 81%, or at least about 82%, or at least about 83%, or atleast about 84%, or at least about 85%, or at least about 86%, or atleast about 87%, or at least about 88%, or at least about 89%, or atleast about 90%, or at least about 91%, or at least about 92%, or atleast about 93%, or at least about 94%, or at least about 95%, or atleast about 96%, or at least about 97%, or at least about 98%, or atleast about 99% sequence identity with the known wild type amino acidsequences of the soluble agents described herein (e.g. about 60%, orabout 61%, or about 62%, or about 63%, or about 64%, or about 65%, orabout 66%, or about 67%, or about 68%, or about 69%, or about 70%, orabout 71%, or about 72%, or about 73%, or about 74%, or about 75%, orabout 76%, or about 77%, or about 78%, or about 79%, or about 80%, orabout 81%, or about 82%, or about 83%, or about 84%, or about 85%, orabout 86%, or about 87%, or about 88%, or about 89%, or about 90%, orabout 91%, or about 92%, or about 93%, or about 94%, or about 95%, orabout 96%, or about 97%, or about 98%, or about 99% sequence identity).

In various embodiments the modified soluble agent comprises an aminoacid sequence that has at least about 60%, or at least about 61%, or atleast about 62%, or at least about 63%, or at least about 64%, or atleast about 65%, or at least about 66%, or at least about 67%, or atleast about 68%, or at least about 69%, or at least about 70%, or atleast about 71%, or at least about 72%, or at least about 73%, or atleast about 74%, or at least about 75%, or at least about 76%, or atleast about 77%, or at least about 78%, or at least about 79%, or atleast about 80%, or at least about 81%, or at least about 82%, or atleast about 83%, or at least about 84%, or at least about 85%, or atleast about 86%, or at least about 87%, or at least about 88%, or atleast about 89%, or at least about 90%, or at least about 91%, or atleast about 92%, or at least about 93%, or at least about 94%, or atleast about 95%, or at least about 96%, or at least about 97%, or atleast about 98%, or at least about 99% sequence identity with any of thesequences disclosed herein (e.g. about 60%, or about 61%, or about 62%,or about 63%, or about 64%, or about 65%, or about 66%, or about 67%, orabout 68%, or about 69%, or about 70%, or about 71%, or about 72%, orabout 73%, or about 74%, or about 75%, or about 76%, or about 77%, orabout 78%, or about 79%, or about 80%, or about 81%, or about 82%, orabout 83%, or about 84%, or about 85%, or about 86%, or about 87%, orabout 88%, or about 89%, or about 90%, or about 91%, or about 92%, orabout 93%, or about 94%, or about 95%, or about 96%, or about 97%, orabout 98%, or about 99% sequence identity).

In various embodiments, the modified soluble agent comprises an aminoacid sequence having one or more amino acid mutations. In someembodiments, the one or more amino acid mutations may be independentlyselected from substitutions, insertions, deletions, and truncations.

In some embodiments, the amino acid mutations are amino acidsubstitutions, and may include conservative and/or non-conservativesubstitutions.

“Conservative substitutions” may be made, for instance, on the basis ofsimilarity in polarity, charge, size, solubility, hydrophobicity,hydrophilicity, and/or the amphipathic nature of the amino acid residuesinvolved. The 20 naturally occurring amino acids can be grouped into thefollowing six standard amino acid groups: (1) hydrophobic: Met, Ala,Val, Leu, Ile; (2) neutral hydrophilic: Cys, Ser, Thr; Asn, Gln; (3)acidic: Asp, Glu; (4) basic: His, Lys, Arg; (5) residues that influencechain orientation: Gly, Pro; and (6) aromatic: Trp, Tyr, Phe.

As used herein, “conservative substitutions” are defined as exchanges ofan amino acid by another amino acid listed within the same group of thesix standard amino acid groups shown above. For example, the exchange ofAsp by Glu retains one negative charge in the so modified polypeptide.In addition, glycine and proline may be substituted for one anotherbased on their ability to disrupt α-helices.

As used herein, “non-conservative substitutions” are defined asexchanges of an amino acid by another amino acid listed in a differentgroup of the six standard amino acid groups (1) to (6) shown above.

In various embodiments, the substitutions may also include non-classicalamino acids (e.g. selenocysteine, pyrrolysine, N-formylmethionineβ-alanine, GABA and δ-Aminolevulinic acid, 4-aminobenzoic acid (PABA),D-isomers of the common amino acids, 2,4-diaminobutyric acid, α-aminoisobutyric acid, 4-aminobutyric acid, Abu, 2-amino butyric acid, γ-Abu,ε-Ahx, 6-amino hexanoic acid, Aib, 2-amino isobutyric acid, 3-aminopropionic acid, ornithine, norleucine, norvaline, hydroxyproline,sarcosme, citrulline, homocitrulline, cysteic acid, t-butylglycine,t-butylalanine, phenylglycine, cyclohexylalanine, β-alanine,fluoro-amino acids, designer amino acids such as β methyl amino acids, Cα-methyl amino acids, N α-methyl amino acids, and amino acid analogs ingeneral).

Illustrative mutations which provide reduced affinity and/or activity(e.g. agonistic) at a therapeutic receptor are found in WO 2013/107791(e.g. with regard to interferons), WO 2015/007542 (e.g. with regard tointerleukins), and WO 2015/007903 (e.g. with regard to TNF), the entirecontents of each of which are hereby incorporated by reference.Illustrative mutations which provide reduced affinity and/or activity(e.g. antagonistic) at a therapeutic receptor are found in WO2015/007520, the entire contents of which are hereby incorporated byreference.

As described herein, the modified soluble agents bear mutations thataffects affinity and/or activity at one or more receptors. In variousembodiments, there is reduced affinity and/or activity at a therapeuticreceptor, e.g. a receptor through which a desired therapeutic effect ismediated (e.g. agonism or antagonism). In various embodiments, there issubstantially reduced or ablated affinity and/or activity at anotherreceptor, e.g. a receptor through which a desired therapeutic effect isnot mediated (e.g. as the result of promiscuity of binding). Thereceptors of any modified soluble agents, e.g. one of the cytokines,growth factors, and hormones as described herein, are known in the art.

In some embodiments, the modified soluble agent comprises one or moremutations that cause the soluble agent to have reduced affinity and/oractivity for a type I cytokine receptor, a type II cytokine receptor, achemokine receptor, a receptor in the Tumor Necrosis Factor Receptor(TNFR) superfamily, TGF-beta Receptors, a receptor in the immunoglobulin(Ig) superfamily, and/or a receptor in the tyrosine kinase superfamily.In such embodiments, any of these receptors may be the therapeuticreceptor.

In some embodiments, the modified soluble agent also comprises one ormore mutations that cause the soluble agent to have substantiallyreduced or ablated affinity and/or activity for a different type Icytokine receptor, type II cytokine receptor, chemokine receptor, areceptor in the Tumor Necrosis Factor Receptor (TNFR) superfamily,TGF-beta receptor, a receptor in the immunoglobulin (Ig) superfamily, ora receptor in the tyrosine kinase superfamily. In such embodiments, anyof these receptors may be the other receptor.

Type I cytokine receptors are known in the art and include, but are notlimited to receptors for IL2 (beta-subunit), IL3, IL4, IL5, IL6, IL7,IL9, IL11, IL12, GM-CSF, G-CSF, LIF, CNTF, and also the receptors forThrombopoietin (TPO), Prolactin, and Growth hormone. Illustrative type Icytokine receptors include, but are not limited to, GM-CSF receptor,G-CSF receptor, LIF receptor, CNTF receptor, TPO receptor, and type I ILreceptors. In various embodiments, the therapeutic receptor is a Type Icytokine receptor. In various embodiments, the other receptor is a TypeI cytokine receptor.

Type II cytokine receptors are multimeric receptors composed ofheterologous subunits, and are receptors mainly for interferons. Thisfamily of receptors includes, but is not limited to, receptors forinterferon-α, interferon-β and interferon-γ, IL10, IL22, and tissuefactor. Illustrative type II cytokine receptors include, but are notlimited to, IFN-α receptor (e.g. IFNAR1 and IFNAR2), IFN-β receptor,IFN-γ receptor (e.g. IFNGR1 and IFNGR2), and type II IL receptors. Invarious embodiments, the therapeutic receptor is a Type II cytokinereceptor. In various embodiments, the other receptor is a Type IIcytokine receptor.

Chemokine receptors are G protein-coupled receptors with seventransmembrane structure and coupled to G-protein for signaltransduction. Chemokine receptors include, but are not limited to, CCchemokine receptors, CXC chemokine receptors, CX3C chemokine receptors,and XC chemokine receptor (XCR1). Illustrative chemokine receptorsinclude, but are not limited to, CCR1, CCR2, CCR3, CCR4, CCR5, CCR6,CCR7, CCR8, CCR9, CCR10, CXCR1, CXCR2, CXCR3, CXCR3B, CXCR4, CXCR5,CSCR6, CXCR7, XCR1, and CX3CR1. In various embodiments, the therapeuticreceptor is a G protein-coupled receptor. In various embodiments, theother receptor is a Type II cytokine G protein-coupled receptor.

Tumor necrosis factor receptor (TNFR) family members share acysteine-rich domain (CRD) formed of three disulfide bonds surrounding acore motif of CXXCXXC creating an elongated molecule. Exemplary tumornecrosis factor receptor family members include: CDI 20a (TNFRSFIA), CD120b (TNFRSFIB), Lymphotoxin beta receptor (LTBR, TNFRSF3), CD 134(TNFRSF4), CD40 (CD40, TNFRSF5), FAS (FAS, TNFRSF6), TNFRSF6B(TNFRSF6B), CD27 (CD27, TNFRSF7), CD30 (TNFRSF8), CD137 (TNFRSF9),TNFRSFIOA (TNFRSFIOA), TNFRSFIOB, (TNFRSFIOB), TNFRSFIOC (TNFRSFIOC),TNFRSFIOD (TNFRSFIOD), RANK (TNFRSFI IA), Osteoprotegerin (TNFRSFI IB),TNFRSF12A (TNFRSF12A), TNFRSF13B (TNFRSF13B), TNFRSF13C (TNFRSF13C),TNFRSF14 (TNFRSF14), Nerve growth factor receptor (NGFR, TNFRSF16),TNFRSF17 (TNFRSF17), TNFRSF18 (TNFRSF18), TNFRSF19 (TNFRSF19), TNFRSF21(TNFRSF21), and TNFRSF25 (TNFRSF25). In various embodiments, thetherapeutic receptor is a TNFR family member. In various embodiments,the other receptor is a TNFR family member. In an embodiment, the TNFRfamily member is CDI 20a (TNFRSFIA) or TNF-R1. In another embodiment,the TNFR family member is CD 120b (TNFRSFIB) or TNF-R2.

TGF-beta receptors are single pass serine/threonine kinase receptors.TGF-beta receptors include, but are not limited to, TGFBR1, TGFBR2, andTGFBR3. In various embodiments, the therapeutic receptor is a TGF-betareceptor. In various embodiments, the other receptor is a TGF-betareceptor.

Receptors in the immunoglobulin (Ig) superfamily share structuralhomology with immunoglobulins. Receptors in the Ig superfamily include,but are not limited to, interleukin-1 receptors, CSF-1R, PDGFR (e.g.PDGFRA and PDGFRB), and SCFR. In various embodiments, the therapeuticreceptor is an Ig superfamily receptor. In various embodiments, theother receptor is an Ig superfamily receptor.

Receptors in the tyrosine kinase superfamily are well known in the art.There are about 58 known receptor tyrosine kinases (RTKs), grouped into20 subfamilies. Receptors in the tyrosine kinase superfamily include,but are not limited to, FGF receptors and their various isoforms such asFGFR1, FGFR2, FGFR3, FGFR4, and FGFR5. In various embodiments, thetherapeutic receptor is a tyrosine kinase superfamily receptor. Invarious embodiments, the other receptor is a tyrosine kinase superfamilyreceptor.

In an embodiment, the modified soluble agent is interferon α. In suchembodiments, the modified soluble agent has reduced affinity and/oractivity for the IFN-α/β receptor (IFNAR) that includes IFNAR1 andIFNAR2 chains. In one embodiment, the modified interferon α has reducedaffinity and/or activity at IFNAR1 and substantially reduced or ablatedaffinity and/or activity at IFNAR2. In one embodiment, the modifiedinterferon α has reduced affinity and/or activity at IFNAR2 andsubstantially reduced or ablated affinity and/or activity at IFNAR1.

Mutant forms of interferon α are known to the person skilled in the art.In an illustrative embodiment, the modified signaling agent is theallelic form human IFN-α2a having the amino acid sequence of:

IFN-α2a (SEQ ID NO: 1):CDLPQTHSLGSRRTLMLLAQMRKISLFSCLKDRHDFGFPQEEFGNQFQKAETIPVLHEMIQQIFNLFSTKDSSAAWDETLLDKFYTELYQQLNDLEACVIQGVGVTETPLMKEDSILAVRKYFQRITLYLKEKKYSPCAWEVVRAEIMRS FSLSTNLQESLRSKE

In an illustrative embodiment, the modified signaling agent is theallelic form human IFN-α2b having the amino acid sequence of (whichdiffers from IFN-α2a at amino acid position 23):

IFN-α2b (SEQ ID NO: 2):CDLPQTHSLGSRRTLMLLAQMRRISLFSCLKDRHDFGFPQEEFGNQFQKAETIPVLHEMIQQIFNLFSTKDSSAAWDETLLDKFYTELYQQLNDLEACVIQGVGVTETPLMKEDSILAVRKYFQRITLYLKEKKYSPCAWEVVRAEIMRS FSLSTNLQESLRSKE

In some embodiments, said IFN-α2 mutant (IFN-α2a or IFN-α2b) is mutatedat one or more amino acids at positions 144-154, such as amino acidpositions 148, 149 and/or 153. In some embodiments, the IFN-α2 mutantcomprises one or more mutations selected from L153A, R149A, and M148A.Such mutants are described, for example, in WO2013/107791 and Piehler etal., (2000) J. Biol. Chem, 275:40425-33, the entire contents of all ofwhich are hereby incorporated by reference.

In some embodiments, the IFN-α2 mutants have reduced affinity and/oractivity for IFNAR1. In some embodiments, the IFN-α2 mutant comprisesone or more mutations selected from F64A, N65A, T69A, L80A, Y85A, andY89A, as described in WO2010/030671, the entire contents of which ishereby incorporated by reference.

In some embodiments, the IFN-α2 mutant comprises one or more mutationsselected from K133A, R144A, R149A, and L153A as described inWO2008/124086, the entire contents of which is hereby incorporated byreference.

In some embodiments, the IFN-α2 mutant comprises one or more mutationsselected from R120E and R120E/K121E, as described in WO2015/007520 andWO2010/030671, the entire contents of which are hereby incorporated byreference. In such embodiments, said IFN-α2 mutant antagonizes wildtypeIFN-α2 activity. In such embodiments, said mutant IFN-α2 has reducedaffinity and/or activity for IFNAR1 while affinity and/or activity ofIFNR2 is retained.

In some embodiments, the human IFN-α2 mutant comprises (1) one or moremutations selected from R120E and R120E/K121E, which, without wishing tobe bound by theory, create an antagonistic effect and (2) one or moremutations selected from K133A, R144A, R149A, and L153A, which, withoutwishing to be bound by theory, allow for an attenuated effect at, forexample, IFNAR2. In an embodiment, the human IFN-α2 mutant comprisesR120E and L153A.

In some embodiments, the human IFN-α2 mutant comprises one or moremutations selected from, L15A, A19W, R22A, R23A, L26A, F27A, L30A, L30V,K31A, D32A, R33K, R33A, R33Q, H34A, D35A, Q40A, D114R, L117A, R120A,R125A, K134A, R144A, A145G, A145M, M148A, R149A, S152A, L153A, and N156Aas disclosed in WO 2013/059885, the entire disclosures of which arehereby incorporated by reference. In some embodiments, the human IFN-α2mutant comprises the mutations H57Y, E58N, Q61S, and/or L30A asdisclosed in WO 2013/059885. In some embodiments, the human IFN-α2mutant comprises the mutations H57Y, E58N, Q61S, and/or R33A asdisclosed in WO 2013/059885. In some embodiments, the human IFN-α2mutant comprises the mutations H57Y, E58N, Q61S, and/or M148A asdisclosed in WO 2013/059885. In some embodiments, the human IFN-α2mutant comprises the mutations H57Y, E58N, Q61S, and/or L153A asdisclosed in WO 2013/059885. In some embodiments, the human IFN-α2mutant comprises the mutations N65A, L80A, Y85A, and/or Y89A asdisclosed in WO 2013/059885. In some embodiments, the human IFN-α2mutant comprises the mutations N65A, L80A, Y85A, Y89A, and/or D114A asdisclosed in WO 2013/059885.

In an embodiment, the modified soluble agent is interferon β. In suchembodiments, the modified soluble agent has reduced affinity and/oractivity for the IFN-α/β receptor (IFNAR) that includes IFNAR1 andIFNAR2 chains. In one embodiment, the modified interferon β has reducedaffinity and/or activity at IFNAR1 and substantially reduced or ablatedaffinity and/or activity at IFNAR2. In one embodiment, the modifiedinterferon β has reduced affinity and/or activity at IFNAR2 andsubstantially reduced or ablated affinity and/or activity at IFNAR1.

In an embodiment, the modified soluble agent is interferon γ. In suchembodiments, the modified soluble agent has reduced affinity and/oractivity for the interferon-gamma receptor (IFNGR) that includes IFNGR1and IFNGR2 chains. In one embodiment, the modified interferon γ hasreduced affinity and/or activity at IFNGR1 and substantially reduced orablated affinity and/or activity at IFNGR2. In one embodiment, themodified interferon γ has reduced affinity and/or activity at IFNGR2 andsubstantially reduced or ablated affinity and/or activity at IFNGR1.

In some embodiments, the modified soluble agent is vascular endothelialgrowth factor (VEGF). In some embodiments, the VEGF is VEGF-A, VEGF-B,VEFG-C, VEGF-D, or VEGF-E and isoforms thereof including the variousisoforms of VEGF-A such as VEGF₁₂₁, VEGF₁₂₁b, VEGF₁₄₅, VEGF₁₆₅,VEGF₁₆₅b, VEGF₁₈₉, and VEGF₂₀₆. In some embodiments, the modifiedsoluble agent has reduced affinity and/or activity for VEGFR-1 (Flt-1)and/or VEGFR-2 (KDR/Flk-1). In an embodiment, the modified soluble agenthas reduced affinity and/or activity for VEGFR-2 (KDR/Flk-1) andsubstantially reduced or ablated affinity and/or activity for VEGFR-1(Flt-1). Such an embodiment finds use in, for example, wound healingmethods or treatment of ischemia-related diseases (without wishing to bebound by theory, mediated by VEGFR-2's effects on endothelial cellfunction and angiogenesis). In various embodiments, VEGFR-1 (Flt-1),which is linked to cancers and pro-inflammatory activities, is avoided.In various embodiments, VEGFR-1 (Flt-1) acts a decoy receptor andtherefore substantially reduced or ablated affinity at this receptoravoids sequestration of the therapeutic agent. In an embodiment, themodified agent has substantially reduced or ablated affinity and/oractivity for VEGFR-1 (Flt-1) and substantially reduced or ablatedaffinity and/or activity for VEGFR-2 (KDR/Flk-1). In some embodiments,the VEGF is VEGF-C or VEGF-D. In such embodiments, the modified solubleagent has reduced affinity and/or activity for VEGFR-3.

VEGF is a potent growth factor that plays major roles in physiologicalbut also pathological angiogenesis, regulates vascular permeability andcan act as a growth factor on cells expressing VEGF receptors.Additional functions include, among others, stimulation of cellmigration in macrophage lineage and endothelial cells. Several membersof the VEGF family of growth factors exist, as well as at least threereceptors (VEGFR-1, VEGFR-2, and VEGFR-3). Members of the VEGF familycan bind and activate more than one VEGFR type. For example, VEGF-Abinds VEGFR-1 and -2, while VEGF-C can bind VEGFR-2 and -3. VEGFR-1 and-2 activation regulates angiogenesis while VEGFR-3 activation isassociated with lymphangiogenesis. The major proangiogenic signal isgenerated from activation of VEGFR-2. VEGFR-1 activation has beenreported to be possibly associated with a negative role in angiogenesis.It has also been reported that VEGFR-1 signaling is important forprogression of tumors in vivo via bone marrow-derived VEGFR-1 positivecells (contributing to formation of premetastatic niche in the bone).Several therapies based on VEGF-A directed/neutralizing therapeuticantibodies have been developed, primarily for use in treatment ofvarious human tumors relying on angiogenesis. These are not without sideeffects. This may not be surprising considering that these operate asgeneral, non-cell/tissue specific VEGFNEGFR interaction inhibitors.Hence, it would be desirable to restrict VEGF (e.g. VEGF-A)/VEGFR-2inhibition to specific target cells (e.g. tumor vasculature endothelialcells).

Proangiogenic therapies are also important in various diseases (e.g.ischemic heart disease, bleeding etc.), and include VEGF-basedtherapeutics. Activation of VEGFR-2 is proangiogenic (acting onendothelial cells). Activation of VEGFR-1 can cause stimulation ofmigration of inflammatory cells (including, for example, macrophages)and lead to inflammation associated hypervascular permeability.Activation of VEFGR-1 can also promote bone marrow associated tumorniche formation. Thus, VEGF based therapeutic selective for VEGFR-2activation would be desirable in this case. In addition, cell specifictargeting, e.g. to endothelial cells, would be desirable.

In some embodiments, the modified soluble agent has reduced affinityand/or activity (e.g. antagonistic) for VEGFR-2 and has substantiallyreduced or ablated affinity and/or activity for VEGFR-1. When targetedto tumor vasculature endothelial cells via a targeting moiety that bindsto a tumor endothelial cell marker (e.g. PSMA and others), suchconstruct inhibits VEGFR-2 activation specifically on suchmarker-positive cells, while not activating VEGFR-1 en route and ontarget cells (if activity ablated), thus eliminating induction ofinflammatory responses, for example. This would provide a more selectiveand safe anti-angiogenic therapy for many tumor types as compared toVEGF-A neutralizing therapies.

In some embodiments, the modified soluble agent has reduced affinityand/or activity (e.g. agonistic) for VEGFR-2 and has substantiallyreduced or ablated affinity and/or activity for VEGFR-1. Throughtargeting to vascular endothelial cells, such construct, in someembodiments, promotes angiogenesis without causing VEGFR-1 associatedinduction of inflammatory responses. Hence, such a construct would havetargeted proangiogenic effects with substantially reduced risk of sideeffects caused by systemic activation of VEGFR-2 as well as VEGFR-1.

In an illustrative embodiment, the modified soluble agent is VEGF₁₆₅,which has the amino acid sequence:

VEGF 165 (wild type) (SEQ ID NO: 3):APMAEGGGQNHHEVVKFMDVYQRSYCHPIETLVDIFQEYPDEIEYIFKPSCVPLMRCGGCCNDEGLECVPTEESNITMQIMRIKPHQGQHIGEMSFLQHNKCECRPKKDRARQENPCGPCSERRKHLFVQDPQTCKCSCKNTDSRCKARQ LELNERTCRCDKPRR

In another illustrative embodiment, the modified soluble agent isVEGF₁₆₅b, which has the amino acid sequence:

VEGF 165b (wild type) (SEQ ID NO: 4):APMAEGGGQNHHEVVKFMDVYQRSYCHPIETLVDIFQEYPDEIEYIFKPSCVPLMRCGGCCNDEGLECVPTEESNITMQIMRIKPHQGQHIGEMSFLQHNKCECRPKKDRARQENPCGPCSERRKHLFVQDPQTCKCSCKNTDSRCKARQ LELNERTCRSLTRKD

In these embodiments, the modified soluble agent has a mutation at aminoacid I83 (e.g., a substitution mutation at 183, e.g., 183K, 183R, or183H). Without wishing to be bound by theory, it is believed that suchmutations may result in reduced receptor binding affinity. See, forexample, U.S. Pat. No. 9,078,860, the entire contents of which arehereby incorporated by reference.

In an embodiment, the modified soluble agent is TNF-α. TNF is apleiotropic cytokine with many diverse functions, including regulationof cell growth, differentiation, apoptosis, tumorigenesis, viralreplication, autoimmunity, immune cell functions and trafficking,inflammation, and septic shock. It binds to two distinct membranereceptors on target cells: TNFR1 (p55) and TNFR2 (p75). TNFR1 exhibits avery broad expression pattern whereas TNFR2 is expressed preferentiallyon certain populations of lymphocytes, Tregs, endothelial cells, certainneurons, microglia, cardiac myocytes and mesenchymal stem cells. Verydistinct biological pathways are activated in response to receptoractivation, although there is also some overlap. As a general rule,without wishing to be bound by theory, TNFR1 signaling is associatedwith induction of apoptosis (cell death) and TNFR2 signaling isassociated with activation of cell survival signals (e.g. activation ofNFkB pathway). Administration of TNF is systemically toxic, and this islargely due to TNFR1 engagement. However, it should be noted thatactivation of TNFR2 is also associated with a broad range of activitiesand, as with TNFR1, in the context of developing TNF based therapeutics,control over TNF targeting and activity is important.

In some embodiments, the modified soluble agent has reduced affinityand/or activity for TNFR1 and/or TNFR2. TNFR1 is expressed in mosttissues, and is involved in cell death signaling while, by contrast,TNFR2 is involved in cell survival signaling. Accordingly in embodimentsdirected to methods of treating cancer, the modified soluble agent hasreduced affinity and/or activity for TNFR1 and substantially reduced orablated affinity and/or activity for TNFR2. In these embodiments, thechimeric proteins may be targeted to a cell for which apoptosis isdesired, e.g. a tumor cell or a tumor vasculature endothelial cell. Inembodiments directed to methods of promoting cell survival, for example,in neurogenesis for the treatment of neurodegenerative disorders, themodified soluble agent has reduced affinity and/or activity for TNFR2and substantially reduced or ablated affinity and/or activity for TNFR1.Stated another way, the present chimeric proteins, in some embodiments,comprise modified TNF-α agent that allows of favoring either death orsurvival signals.

In some embodiments, the chimeric protein has a modified TNF havingreduced affinity and/or activity for TNFR1 and substantially reduced orablated affinity and/or activity for TNFR2. Such a chimera, in someembodiments, is a more potent inducer of apoptosis as compared to a wildtype TNF and/or a chimera bearing only mutation(s) causing reducedaffinity and/or activity for TNFR1. Such a chimera, in some embodiments,find use in inducing tumor cell death or a tumor vasculature endothelialcell death (e.g. in the treatment of cancers). Also, in someembodiments, these chimeras avoid or reduce activation of T_(reg) cellsvia TNFR2, for example, thus further supporting TNFR1-mediated antitumoractivity in vivo.

In some embodiments, the chimeric protein has a modified TNF havingreduced affinity and/or activity for TNFR2 and substantially reduced orablated affinity and/or activity for TNFR1. Such a chimera, in someembodiments, is a more potent activator of cell survival in some celltypes, which may be a specific therapeutic objective in various diseasesettings, including without limitation, stimulation of neurogenesis. Inaddition, such a TNFR2-favoring chimeras also are useful in thetreatment of autoimmune diseases (e.g. Crohn's, diabetes, MS, colitisetc. and many others described herein). In some embodiments, the chimeraare targeted to auto-reactive T cells. In some embodiments, the chimerapromotes T_(reg) cell activation and indirect suppression of cytotoxic Tcells.

In some embodiments, the chimera causes the death of auto-reactive Tcells, e.g. by activation of TNFR2 and avoidance TNFR1 (e.g. a modifiedTNF having reduced affinity and/or activity for TNFR2 and substantiallyreduced or ablated affinity and/or activity for TNFR1). Without wishingto be bound by theory, these auto-reactive T cells, e.g. by NFkBlesions, have their apoptosis/survival signals reversed.

In some embodiments, a TNFR-2 focused chimera has additional therapeuticapplications in diseases, including various autoimmune diseases, heartdisease, de-myelinating and neurodegenerative disorders, and infectiousdisease, among others.

In an embodiment, the wild type TNF-α has the amino acid sequence of:

TNF-α (wild type) (SEQ ID NO: 5):VRSSSRTPSDKPVAHVVANPQAEGQLQWLNRRANALLANGVELRDNQLVVPSEGLYLIYSQVLFKGQGCPSTHVLLTHTISRIAVSYQTKVNLLSAIKSPCQRETPEGAEAKPWYEPIYLGGVFQLEKGDRLSAEINRPDYLDFAESGQV YFGIIAL

In such embodiments, the modified soluble agent has mutations at one ormore amino acid positions 29, 31, 32, 84, 85, 86, 87, 88, 89, 145, 146and 147 which produces a modified TNF-α with reduced receptor bindingaffinity. See, for example, U.S. Pat. No. 7,993,636, the entire contentsof which are hereby incorporated by reference.

In some embodiments, the modified human TNF-α moiety has mutations atone or more amino acid positions R32, N34, Q67, H73, L75, T77, S86, Y87,V91, I97, T105, P106, A109, P113, Y115, E127, N137, D143, A145, and E146as described, for example, in WO/2015/007903, the entire contents ofwhich is hereby incorporated by reference (numbering according to thehuman TNF sequence, Genbank accession number BAG70306, versionBAG70306.1 GI: 197692685). In some embodiments, the modified human TNF-αmoiety has substitution mutations selected from L29S, R32G, R32W, N34G,Q67G, H73G, L75G, L75A, L75S, T77A, S86G, S86T, Y87Q, Y87L, Y87A, Y87F,Y87H, V91G, V91A, 197A, 197Q, 197S, T105G, P106G, A109Y, P113G, Y115G,Y115A, E127G, N137G, D143N, A145G, A145R, A145T, E146D, E146K, andS147D. In an embodiment, the human TNF-α moiety has a mutation selectedfrom Y87Q, Y87L, Y87A, Y87F, and Y87H. In another embodiment, the humanTNF-α moiety has a mutation selected from I97A, I97Q, and I97S. In afurther embodiment, the human TNF-α moiety has a mutation selected fromY115A and Y115G. In an embodiment, the human TNF-α moiety has an E146Kmutation. In an embodiment, the human TNF-α moiety has an Y87H and anE146K mutation. In an embodiment, the human TNF-α moiety has an Y87H andan A145R mutation. In an embodiment, the human TNF-α moiety has a R32Wand a S86T mutation. In an embodiment, the human TNF-α moiety has a R32Wand an E146K mutation. In an embodiment, the human TNF-α moiety has aL29S and a R32W mutation. In an embodiment, the human TNF-α moiety has aD143N and an A145R mutation. In an embodiment, the human TNF-α moietyhas a D143N and an A145R mutation. In an embodiment, the human TNF-αmoiety has an A145T, an E146D, and a S147D mutation. In an embodiment,the human TNF-α moiety has an A145T and a S147D mutation.

In some embodiments, the modified TNF-α agent has one or more mutationsselected from N39Y, S147Y, and Y87H, as described in WO2008/124086, theentire contents of which is hereby incorporated by reference.

In some embodiments, the modified TNF-α has one or more amino acidmutations disclosed herein with reference to SEQ ID NO: 5.

In some embodiments, the present chimeric protein comprises a targetingmoiety, such as a heavy-chain-only antibody (VHH) directed against CD20,e.g. human CD20 and a modified soluble agent is a TNF having about 90%,or about 93%, or about 95%, or about 97%, or about 98% identity with theamino acid sequence of SEQ ID NO: 5, as well as one or more (e.g. twoof) of the following mutations: L29X, R32X, S86X, Y87X, D143X, A145X,E146X, and S147X, where X is any amino acid, such as (1) hydrophobic:Met, Ala, Val, Leu, Ile; (2) neutral hydrophilic: Cys, Ser, Thr; Asn,Gln; (3) acidic: Asp, or Glu; (4) basic: His, Lys, Arg; (5) residuesthat influence chain orientation: Gly, Pro; and (6) aromatic: Trp, Tyr,Phe. In any of these embodiments, the TNF of having about 90%, or about93%, or about 95%, or about 97%, or about 98% identity with the aminoacid sequence of SEQ ID NO: 5 is a trimeric single polypeptide chainconstruct.

In some embodiments, the present chimeric protein comprises a targetingmoiety, such as a heavy-chain-only antibody (VHH) directed against CD20,e.g. human CD20 and a modified soluble agent is a TNF having about 90%,or about 93%, or about 95%, or about 97%, or about 98% identity with theamino acid sequence of SEQ ID NO: 5 as well as one or more of thefollowing mutations: L29X, where X is neutral hydrophilic amino acidselected from Cys, Ser, Thr; Asn, Gln; R32X, where X is an aromaticamino acid selected from Trp, Tyr, Phe; S86X, where X is a neutralhydrophilic amino acid selected from Cys, Ser, Thr; Asn, Gln; Y87X,where X is hydrophobic: Met, Ala, Val, Leu, Ile, or a basic amino acidselected from His, Lys, Arg, or an aromatic amino acid selected fromTrp, Tyr, Phe; D143X, where X is a neutral hydrophilic amino acidselected from Cys, Ser, Thr; Asn, Gln; A145X, where X is a neutralhydrophilic amino acid selected from Cys, Ser, Thr; Asn, Gln or a basicamino acid selected from His, Lys, Arg; E146X, where X an acidic aminoacid selected from Asp or Glu or a basic amino acid selected from His,Lys, or Arg; and S147X, where X is an acidic amino acid selected fromAsp or Glu. In any of these embodiments, the TNF of having about 90%, orabout 93%, or about 95%, or about 97%, or about 98% identity with theamino acid sequence of SEQ ID NO: 5 is a trimeric single polypeptidechain construct.

In some embodiments, the present chimeric protein comprises a targetingmoiety, such as a heavy-chain-only antibody (VHH) directed against CD20,e.g. human CD20 and a modified soluble agent is a TNF having about 90%,or about 93%, or about 95%, or about 97%, or about 98% identity with theamino acid sequence of SEQ ID NO: 5 as well as one or more of thefollowing mutations: L29S, R32W, S86T, Y87A, H, or F, D143N, A145R or T,E146D or K, S147D. In any of these embodiments, the TNF of having about90%, or about 93%, or about 95%, or about 97%, or about 98% identitywith the amino acid sequence of SEQ ID NO: 5 is a trimeric singlepolypeptide chain construct.

In some embodiments, the present chimeric protein comprises a targetingmoiety, such as a heavy-chain-only antibody (VHH) directed against CD20,e.g. human CD20 and a modified soluble agent is a TNF having about 90%,or about 93%, or about 95%, or about 97%, or about 98% identity with theamino acid sequence of SEQ ID NO: 5 as well as one or more of thefollowing mutations: Y87H and E146K. In any of these embodiments, theTNF of having about 90%, or about 93%, or about 95%, or about 97%, orabout 98% identity with the amino acid sequence of SEQ ID NO: 5 is atrimeric single polypeptide chain construct.

In some embodiments, the present chimeric protein comprises a targetingmoiety, such as a heavy-chain-only antibody (VHH) directed against CD20,e.g. human CD20 and a modified soluble agent is a TNF having about 90%,or about 93%, or about 95%, or about 97%, or about 98% identity with theamino acid sequence of SEQ ID NO: 5 as well as one or more of thefollowing mutations: R32W and S86T. In any of these embodiments, the TNFof having about 90%, or about 93%, or about 95%, or about 97%, or about98% identity with the amino acid sequence of SEQ ID NO: 5 is a trimericsingle polypeptide chain construct.

In some embodiments, the present chimeric protein comprises a targetingmoiety, such as a heavy-chain-only antibody (VHH) directed against CD20,e.g. human CD20 and a modified soluble agent is a TNF having about 90%,or about 93%, or about 95%, or about 97%, or about 98% identity with theamino acid sequence of SEQ ID NO: 5 as well as one or more of thefollowing mutations: Y87H and A145R. In any of these embodiments, theTNF of having about 90%, or about 93%, or about 95%, or about 97%, orabout 98% identity with the amino acid sequence of SEQ ID NO: 5 is atrimeric single polypeptide chain construct.

In an embodiment, the modified soluble agent is TNF-β. TNF-β can form ahomotrimer or a heterotrimer with LT-β (LT-α1β2). In some embodiments,the modified soluble agent has substantially reduced or ablated affinityand/or activity for TNFR1 and/or TNFR2 and/or herpes virus entrymediator (HEVM) and/or LT-βR.

In an embodiment, the wild type TNF-β has the amino acid sequence of:

TNF-beta (wild type) (SEQ ID NO: 6):LPGVGLTPSAAQTARQHPKMHLAHSNLKPAAHLIGDPSKQNSLLWRANTDRAFLQDGFSLSNNSLLVPTSGIYFVYSQVVFSGKAYSPKATSSPLYLAHEVQLFSSQYPFHVPLLSSQKMVYPGLQEPWLHSMYHGAAFQLTQGDQLSTH TDGIPHLVLSPSTVFFGAFAL

In such embodiments, the modified soluble agent may comprise mutationsat one or more amino acids at positions 106-113, which produce amodified TNF-β with reduced receptor binding affinity to TNFR2. In anembodiment, the modified soluble agent has one or more substitutionmutations at amino acid positions 106-113. In illustrative embodiments,the substitution mutations are selected from Q107E, Q107D, S106E, S106D,Q107R, Q107N, Q107E/S106E, Q107E/S106D, Q107D/S106E, and Q107D/S106D. Inanother embodiment, the modified soluble agent has an insertion of about1 to about 3 amino acids at positions 106-113.

In some embodiments, the modified agent is a TNF family member (e.g.TNF-alpha, TNF-beta) which can be a single chain trimeric version asdescribed in WO 2015/00790, the entire contents of which areincorporated by reference.

In some embodiments, the modified agent is a TNF family member (e.g.TNF-alpha, TNF-beta) which has reduced affinity and/or activity, i.e.antagonistic activity (see, e.g., WO 2015/007520, the entire contents ofwhich are hereby incorporated by reference) at TNFR1. In theseembodiments, the modified agent is a TNF family member (e.g. TNF-alpha,TNF-beta) which also, optionally, has substantially reduced or ablatedaffinity and/or activity for TNFR2. In some embodiments, the modifiedagent is a TNF family member (e.g. TNF-alpha, TNF-beta) which hasreduced affinity and/or activity, i.e. antagonistic activity (see, e.g.,WO 2015/007520, the entire contents of which are hereby incorporated byreference) at TNFR2. In these embodiments, the modified agent is a TNFfamily member (e.g. TNF-alpha, TNF-beta) which also, optionally, hassubstantially reduced or ablated affinity and/or activity for TNFR1. Theconstructs of such embodiments find use in, for example, methods ofdampening TNF response in a cell specific manner.

In an embodiment, the modified signaling agent is TRAIL. In someembodiments, the modified TRAIL agent has reduced affinity and/oractivity for DR4 (TRAIL-RI) and/or DR5 (TRAIL-RII) and/or DcR1 and/orDcR2. In some embodiments, the modified TRAIL agent has substantiallyreduced or ablated affinity and/or activity for DR4 (TRAIL-RI) and/orDR5 (TRAIL-RII) and/or DcR1 and/or DcR2.

In an embodiment, the wild type TRAIL has the amino acid sequence of:

TRAIL (SEQ ID NO: 7): MAMMEVQGGPSLGQTCVLIVIFTVLLQSLCVAVTYVYFTNELKQMQDKYSKSGIACFLKEDDSYWDPNDEESMNSPCWQVKWQLRQLVRKMILRTSEETISTVQEKQQNISPLVRERGPQRVAAHITGTRGRSNTLSSPNSKNEKALGRKINSWESSRSGHSFLSNLHLRNGELVIHEKGFYYIYSQTYFRFQEEIKENTKNDKQMVQYIYKYTSYPDPILLMKSARNSCWSKDAEYGLYSIYQGGIFELKENDRIFVSVTNEHLIDMDHEASFFGAFLVG

In such embodiments, the modified TRAIL agent may comprise a mutation atamino acid positions T127-R132, E144-R149, E155-H161, Y189-Y209,T214-1220, K224-A226, W231, E236-L239, E249-K251, T261-H264 andH270-E271 (Numbering based on the human sequence, Genbank accessionnumber NP_003801, version 10 NP_003801.1, GI: 4507593; see above). In anembodiment, the modified soluble agent is TGFα. In such embodiments, themodified soluble agent has reduced affinity and/or activity for theepidermal growth factor receptor (EGFR).

In an embodiment, the modified soluble agent is TGFβ. In suchembodiments, the modified soluble agent has reduced affinity and/oractivity for TGFBR1 and/or TGFBR2 and substantially reduced or ablatedaffinity and/or activity for TGFBR3 which, without wishing to be boundby theory, may act as a reservoir of ligand for TGF-beta receptors. Insome embodiments, the TGF may favor TGFBR1 over TGFBR2 or TGFBR2 overTGFBR1. Similarly, LAP, without wishing to be bound by theory, may actas a reservoir of ligand for TGF-beta receptors. In some embodiments,the modified soluble agent has reduced affinity and/or activity forTGFBR1 and/or TGFBR2 and substantially reduced or ablated affinityand/or activity for Latency Associated Peptide (LAP). In someembodiments, such chimeras find use in Camurati-Engelmann disease, orother diseases associated with inappropriate TGFβ signaling.

In some embodiments, the modified agent is a TGF family member (e.g.TGFα, TGFβ) which has reduced affinity and/or activity, i.e.antagonistic activity at one or more of TGFBR1, TGFBR2, TGFBR3. In theseembodiments, the modified agent is a TGF family member (e.g. TGFα, TGFβ)which also, optionally, has substantially reduced or ablated affinityand/or activity at one or more of TGFBR1, TGFBR2, TGFBR3.

In some embodiments, the modified agent is a TGF family member (e.g.TGFα, TGFβ) which has reduced affinity and/or activity, i.e.antagonistic activity at TGFBR1 and/or TGFBR2. In these embodiments, themodified agent is a TGF family member (e.g. TGFα, TGFβ) which also,optionally, has substantially reduced or ablated affinity and/oractivity at TGFBR3.

In an embodiment, the modified soluble agent is IL-1. In an embodiment,the modified soluble agent is IL-1α or IL-1β. In such an embodiment, themodified soluble agent has reduced affinity and/or activity for IL-1R1and/or IL-1RAcP. In some embodiments, the modified soluble agent hassubstantially reduced or ablated affinity and/or activity for IL-1R2.For instance, in some embodiments, the present modified IL-1 agentsavoid interaction at IL-1R2 and therefore substantially reduce itsfunction as a decoy and/or sink for therapeutic agents.

In an embodiment, the wild type IL-1β has the amino acid sequence of:

IL-1 beta (mature form, wild type) (SEQ ID NO: 8):APVRSLNCTLRDSQQKSLVMSGPYELKALHLQGQDMEQQVVFSMSFVQGEESNDKIPVALGLKEKNLYLSCVLKDDKPTLQLESVDPKNYPKKKMEKRFVFNKIEINNKLEFESAQFPNWYISTSQAENMPVFLGGTKGGQDITDFTMQF VSS

IL1 is a proinflammatory cytokine and an important immune systemregulator. It is a potent activator of CD4 T cell responses, increasesproportion of Th17 cells and expansion of IFNγ and IL-4 producing cells.IL-1 is also a potent regulator of CD8⁺ T cells, enhancingantigen-specific CD8⁺ T cell expansion, differentiation, migration toperiphery and memory. IL-1 receptors comprise IL-1RI and IL-1RII.Binding to and signaling through the IL-1RI constitutes the mechanismwhereby IL-1 mediates many of its biological (and pathological)activities. IL1-RII can function as a decoy receptor, thereby reducingIL-1 availability for interaction and signaling through the IL-1RI.

In some embodiments, the modified IL-1 has reduced affinity and/oractivity (e.g. agonistic activity) for IL-1R1 and substantially reducedor ablated affinity and/or activity for IL-1RII. In such embodiments,there is restorable IL-1/IL-1RI signaling and prevention of loss oftherapeutic chimeras at IL-RII and therefore a reduction in dose of IL-1that is required (e.g. relative to wild type or a chimera bearing onlyan attenuation mutation for IL-RI). Such constructs find use in, forexample, methods of treating cancer, including, for example, stimulatingthe immune system to mount an anti-cancer response.

In some embodiments, the modified IL-1 has reduced affinity and/oractivity (e.g. antagonistic activity) for IL-1R1 and substantiallyreduced or ablated affinity and/or activity for IL-1RII. In suchembodiments, there is restorable IL-1/IL-1RI signaling and prevention ofloss of therapeutic chimeras at IL-RII and therefore a reduction in doseof IL-1 that is required (e.g. relative to wild type or a chimerabearing only an attenuation mutation for IL-RI). Such constructs finduse in, for example, methods of treating autoimmune diseases, including,for example, suppressing the immune system.

In such embodiments, the modified signaling agent has a deletion ofamino acids 52-54 which produces a modified human IL-β with reducedbinding affinity for type I IL-1R and reduced biological activity. See,for example, WO 1994/000491, the entire contents of which are herebyincorporated by reference. In some embodiments, the modified human IL-1βhas one or more substitution mutations selected from A117G/P118G, R120X,L122A, T125G/L126G, R127G, Q130X, Q131G, K132A, S137G/Q138Y, L145G,H146X, L145A/L147A, Q148X, Q148G/Q150G, Q150G/D151A, M152G, F162A,F162A/Q164E, F166A, Q164E/E167K, N169G/D170G, I172A, V174A, K208E,K209X, K209A/K210A, K219X, E221X, E221 S/N224A, N224S/K225S, E244K,N245Q (where X can be any change in amino acid, e.g., a non-conservativechange), which exhibit reduced binding to IL-1R, as described, forexample, in WO2015/007542 and WO/2015/007536, the entire contents ofwhich is hereby incorporated by reference (numbering base on the humanIL-1 β sequence, Genbank accession number NP_000567, versionNP-000567.1, GI: 10835145). In some embodiments, the modified humanIL-1β may have one or more mutations selected from R120A, R120G, Q130A,Q130W, H146A, H146G, H146E, H146N, H146R, Q148E, Q148G, Q148L, K209A,K209D, K219S, K219Q, E221S and E221K. In an embodiment, the modifiedhuman IL-1β comprises the mutations Q131G and Q148G. In an embodiment,the modified human IL-1β comprises the mutations Q148G and K208E. In anembodiment, the modified human IL-1β comprises the mutations R120G andQ131G. In an embodiment, the modified human IL-1β comprises themutations R120G and H146G. In an embodiment, the modified human IL-1βcomprises the mutations R120G and K208E. In an embodiment, the modifiedhuman IL-1β comprises the mutations R120G, F162A, and Q164E.

In an embodiment, the modified soluble agent is IL-2. In such anembodiment, the modified soluble agent has reduced affinity and/oractivity for IL-2Rα and/or IL-2Rβ and/or IL-2Rγ. In some embodiments,the modified soluble agent has reduced affinity and/or activity forIL-2Rβ and/or IL-2Rγ. In some embodiments, the modified soluble agenthas substantially reduced or ablated affinity and/or activity forIL-2Rα. Such embodiments may be relevant for treatment of cancer, forinstance when the modified IL-2 is agonistic at IL-2Rβ and/or IL-2Rγ.For instance, the present constructs may favor attenuated activation ofCD8⁺ T cells (which can provide an anti-tumor effect), which have β andγ and disfavor T_(regs) (which can provide an immune suppressive,pro-tumor effect), which have α, β, and γ. Further, in some embodiments,the preferences for IL-2Rβ and/or IL-2Rγ over IL-2Rα avoid IL-2 sideeffects such as pulmonary edema. Also, IL-2-based chimeras are usefulfor the treatment of autoimmune diseases, for instance when the modifiedIL-2 is antagonistic at IL-2Rβ and/or IL-2Rγ. For instance, the presentconstructs may favor attenuated suppression of CD8⁺ T cells (andtherefore dampen the immune response), which have β and γ and disfavorT_(regs) which have α, β, and γ. Alternatively, in some embodiments, thechimeras bearing IL-2 favor the activation of T_(regs), and thereforeimmune suppression, and activation of disfavor of CD8⁺ T cells. Forinstance, these constructs find use in the treatment of diseases ordiseases that would benefit from immune suppression, e.g. autoimmunedisorders.

In some embodiments, the chimeric protein has targeting moieties asdescribed herein the direct to CD8⁺ T cells as well as an IL-2 havingreduced affinity and/or activity for IL-2β and/or IL-2Rγ andsubstantially reduced or ablated affinity and/or activity for IL-2Rα. Insome embodiments, these constructs provide targeted CD8⁺ T cell activityand are generally inactive (or have substantially reduced activity)towards T_(reg) cells. In some embodiments, such constructs haveenhanced immune stimulatory effect compared to wild type IL-2 (e.g.,without wishing to be bound by theory, by not stimulating Tregs), whilsteliminating or reducing the systemic toxicity associated with IL-2.

In an embodiment, the wild type IL-2 has the amino acid sequence of:

IL-2 (mature form, wild type) (SEQ ID NO: 9):APTSSSTKKTQLQLEHLLLDLQMILNGINNYKNPKLTRMLTFKFYMPKKATELKHLQCLEEELKPLEEVLNLAQSKNFHLRPRDLISNINVIVLELKGSETTFMCEYADETATIVEFLNRWITFCQSIISTLT

In such embodiments, the modified soluble agent has one or moremutations at amino acids L72 (L72G, L72A, L72S, L72T, L72Q, L72E, L72N,L72D, L72R, or L72K), F42 (F42A, F42G, F42S, F42T, F42Q, F42E, F42N,F42D, F42R, or F42K) and Y45 (Y45A, Y45G, Y45S, Y45T, Y45Q, Y45E, Y45N,Y45D, Y45R or Y45K). Without wishing to be bound by theory, it isbelieved that these modified IL-2 agents have reduced affinity for thehigh-affinity IL-2 receptor and preserves affinity to theintermediate-affinity IL-2 receptor, as compared to the wild-type IL-2.See, for example, US 20120244112, the entire contents of which arehereby incorporated by reference.

In an embodiment, the modified soluble agent is IL-3. In such anembodiment, the modified soluble agent has reduced affinity and/oractivity for the IL-3 receptor, which is a heterodimer with a uniquealpha chain paired with the common beta (beta c or CD131) subunit.

In an embodiment, the modified soluble agent is IL-4. In such anembodiment, the modified soluble agent has reduced affinity and/oractivity for type 1 and/or type 2 IL-4 receptors. Type 1 IL-4 receptorsare composed of the IL-4Rα subunit with a common γ chain andspecifically bind IL-4. Type 2 IL-4 receptors include an IL-4Rα subunitbound to a different subunit known as IL-13Rα1. In some embodiments, themodified soluble agent has substantially reduced or ablated affinityand/or activity the type 2 IL-4 receptors.

In an embodiment, the wild type IL-4 has the amino acid sequence of:

IL-4 (mature form, wild type) (SEQ ID NO: 10):HKCDITLQEIIKTLNSLTEQKTLCTELTVTDIFAASKNTTEKETFCRAATVLRQFYSHHEKDTRCLGATAQQFHRHKQLIRFLKRLDRNLWGLAGLNSCPVKEANQSTLENFLERLKTIMREKYSKCSS

In such embodiments, the modified soluble agent has one or moremutations at amino acids R121 (R121A, R121D, R121E, R121F, R121H, R1211,R121K, R121N, R121P, R121T, R121W), E122 (E122F), Y124 (Y124A, Y124Q,Y124R, Y124S, Y124T) and S125 (S125A). Without wishing to be bound bytheory, it is believed that these modified IL-4 agents maintain theactivity mediated by the type I receptor, but significantly reduces thebiological activity mediated by the other receptors. See, for example,U.S. Pat. No. 6,433,157, the entire contents of which are herebyincorporated by reference.

In an embodiment, the modified soluble agent is IL-6. IL-6 signalsthrough a cell-surface type I cytokine receptor complex including theligand-binding IL-6R chain (CD126), and the signal-transducing componentgp130. IL-6 may also bind to a soluble form of IL-6R (sIL-6R), which isthe extracellular portion of IL-6R. The sIL-6R/IL-6 complex may beinvolved in neurites outgrowth and survival of neurons and, hence, maybe important in nerve regeneration through remyelination. Accordingly,in some embodiments, the modified soluble agent has reduced affinityand/or activity for IL-6R/gp130 and/or sIL-6R. In some embodiments, themodified soluble agent has substantially reduced or ablated affinityand/or activity for IL-6R/gp130 and/or sIL-6R.

In an embodiment, the wild type IL-6 has the amino acid sequence of:

IL-6 (mature form, wild type) (SEQ ID NO: 11):APVPPGEDSKDVAAPHRQPLTSSERIDKQIRYILDGISALRKETCNKSNMCESSKEALAENNLNLPKMAEKDGCFQSGFNEETCLVKIITGLLEFEVYLEYLQNRFESSEEQARAVQMSTKVLIQFLQKKAKNLDAITTPDPTTNASLTTKLQAQNQWLQDMTTHLILRSFKEFLQSSLRALRQM

In such embodiments, the modified soluble agent has one or moremutations at amino acids 58, 160, 163, 171 or 177. Without wishing to bebound by theory, it is believed that these modified IL-6 agents exhibitreduced binding affinity to IL-6Ralpha and reduced biological activity.See, for example, WO 97/10338, the entire contents of which are herebyincorporated by reference.

In an embodiment, the modified soluble agent is IL-10. In such anembodiment, the modified soluble agent has reduced affinity and/oractivity for IL-10 receptor-1 and IL-10 receptor-2. In some embodiments,the modified soluble agent has substantially reduced affinity and/oractivity for IL-10 receptor-1 and IL-10 receptor-2

In an embodiment, the modified soluble agent is IL-11. In such anembodiment, the modified soluble agent has reduced affinity and/oractivity for IL-11Rα and/or IL-11Rβ and/or gp130. In such an embodiment,the modified soluble agent has substantially or ablated reduced affinityand/or activity for IL-11Rα and/or IL-11Rβ and/or gp130.

In an embodiment, the modified soluble agent is IL-13. In such anembodiment, the modified soluble agent has reduced affinity and/oractivity for the IL-4 receptor (IL-4Rα) and IL-13Rα1. In someembodiments, the modified soluble agent has substantially reduced orablated affinity and/or activity for IL-4 receptor (IL-4Rα) or IL-13Rα1.

In an embodiment, the wild type IL-13 has the amino acid sequence of:

IL-13 (mature form, wild type) (SEQ ID NO: 12):SPGPVPPSTALRELIEELVNITQNQKAPLCNGSMVWSINLTAGMYCAALESLINVSGCSAIEKTQRMLSGFCPHKVSAGQFSSLHVRDTKIEVAQFVKDL LLHLKKLFREGRFN

In such embodiments, the modified soluble agent has one or moremutations at amino acids 13, 16, 17, 66, 69, 99, 102, 104, 105, 106,107, 108, 109, 112, 113 and 114. Without wishing to be bound by theory,it is believed that these modified IL-13 agents exhibit reducedbiological activity. See, for example, WO 2002/018422, the entirecontents of which are hereby incorporated by reference.

In an embodiment, the modified soluble agent is IL-18. In such anembodiment, the modified soluble agent has reduced affinity and/oractivity for IL-18Rα and/or IL-18Rβ. In some embodiments, the modifiedsoluble agent has substantially reduced or ablated affinity and/oractivity for IL-18Rα type II, which is an isoform of IL-18Rα that lacksthe TIR domain required for signaling.

In an embodiment, the modified soluble agent is IL-33. In such anembodiment, the modified soluble agent has reduced affinity and/oractivity for the ST-2 receptor and IL-1RAcP. In such an embodiment, themodified soluble agent has substantially reduced or ablated affinityand/or activity for the ST-2 receptor and IL-1RAcP.

In an embodiment, the modified soluble agent is epidermal growth factor(EGF). EGF is a member of a family of potent growth factors. Membersinclude EGF, HB-EGF, and others such as TGFalpha, amphiregulin,neuregulins, epiregulin, and betacellulin. EGF family receptors includeEGFR (ErbB1), ErbB2, ErbB3 and ErbB4. These may function as homodimericand/or heterodimeric receptor subtypes. The different EGF family membersexhibit differential selectivity for the various receptor subtypes. Forexample, EGF associates with ErbB1/ErbB1, ErbB1/ErbB2, ErbB4/ErbB2 andsome other heterodimeric subtypes. HB-EGF has a similar pattern,although it also associates with ErbB4/4. Modulation of EGF (EGF-like)growth factor signaling, positively or negatively, is of considerabletherapeutic interest. For example, inhibition of EGFRs signaling is ofinterest in the treatment of various cancers where EGFR signalingconstitutes a major growth promoting signal. Alternatively, stimulationof EGFRs signaling is of therapeutic interest in, for example, promotingwound healing (acute and chronic), oral mucositis (a major side-effectof various cancer therapies, including, without limitation radiationtherapy).

In some embodiments, the modified soluble agent has reduced affinityand/or activity for ErbB1, ErbB2, ErbB3, and/or ErbB4. Such embodimentsfind use, for example, in methods of treating wounds. In someembodiments, the modified soluble agent binds to one or more ErbB1,ErbB2, ErbB3, and ErbB4 and antagonizes the activity of the receptor. Insuch embodiments, the modified soluble agent has reduced affinity and/oractivity for ErbB1, ErbB2, ErbB3, and/or ErbB4 which allows for theactivity of the receptor to be antagonized in an attenuated fashion.Such embodiments find use in, for example, treatments of cancer. In anembodiment, the modified soluble agent has reduced affinity and/oractivity for ErbB1. ErbB1 is the therapeutic target of kinaseinhibitors—most have side effects because they are not very selective(e.g., gefitinib, erlotinib, afatinib, brigatinib and icotinib). In someembodiments, attenuated antagonistic ErbB1 signaling is more on-targetand has less side effects than other agents targeting receptors for EGF.

In some embodiments, the modified soluble agent has reduced affinityand/or activity (e.g. antagonistic) for ErbB1 and substantially reducedor ablated affinity and/or activity for ErbB4 or other subtypes it mayinteract with. Through specific targeting via the targeting moiety,cell-selective suppression (antagonism) of ErbB1/ErbB1 receptoractivation would be achieved—while not engaging other receptor subtypespotentially associated with inhibition-associated side effects. Hence,in contrast to EGFR kinase inhibitors, which inhibit EGFR activity inall cell types in the body, such a construct would provide acell-selective (e.g., tumor cell with activated EGFR signaling due toamplification of receptor, overexpression etc.) anti-EGFR (ErbB1) drugeffect with reduced side effects.

In some embodiments, the modified soluble agent has reduced affinityand/or activity (e.g. agonistic) for ErbB4 and/or other subtypes it mayinteract with. Through targeting to specific target cells through thetargeting moiety, a selective activation of ErbB1 signaling is achieved(e.g. epithelial cells). Such a construct finds use, in someembodiments, in the treatment of wounds (promoting would healing) withreduced side effects, especially for treatment of chronic conditions andapplication other than topical application of a therapeutic (e.g.systemic wound healing).

In an embodiment, the modified soluble agent is insulin or insulinanalogs. In such an embodiment, the modified insulin or insulin analoghas reduced affinity and/or activity for the insulin receptor and/orIGF1 or IGF2 receptor. Attenuated response at the insulin receptorallows for the control of diabetes, obesity, metabolic disorders and thelike while directing away from IGF1 or IGF2 receptor avoids pro-cancereffects.

In an embodiment, the modified soluble agent is insulin-like growthfactor-I or insulin-like growth factor-II (IGF-1 or IGF-2). In anembodiment, the modified soluble agent is IGF-1. In such an embodiment,the modified soluble agent has reduced affinity and/or activity for theinsulin receptor and/or IGF1 receptor. In an embodiment, the modifiedsoluble agent may bind to the IGF1 receptor and antagonize the activityof the receptor. In such an embodiment, the modified soluble agent hasreduced affinity and/or activity for IGF1 receptor which allows for theactivity of the receptor to be antagonized in an attenuated fashion. Insuch an embodiment, the modified soluble agent has reduced affinityand/or activity for IGF2 receptor which allows for the activity of thereceptor to be antagonized in an attenuated fashion. In an embodiment,the modified soluble agent has substantially reduced or ablated affinityand/or activity for the insulin receptor and accordingly not interferewith insulin signaling. In various embodiments, this applies to cancertreatment. In various embodiments, the present agents may prevent IRisoform A from causing resistance to cancer treatments.

In an embodiment, the modified soluble agent is erythropoietin (EPO). Inhumans, erythropoietin (EPO) is a 35 kD glycoprotein hormone involved inregulating red blood cell production in the bone marrow. EPO stimulatesthe division and differentiation of committed erythroid progenitor andprecursor cells in the bone marrow and exerts its biological activityby, for instance, binding to receptors on erythroid cells.

Erythropoietin or EPO is a protein that is encoded by the EPO gene, or anatural or engineered variant, family-member, orthologue, fragment orfusion construct thereof. In various embodiments, the EPO proteinincludes by way of non-limiting example, human erythropoietin, mouseerythropoietin, an erythropoietin binding domain, or an erythropoietinfusion protein. In an embodiment, the EPO protein is human EPO. In anembodiment, the human EPO has the amino acid sequence of (the signalpeptide is underlined) (SEQ ID NO: 13):

MGVHECPAWLWLLLSLLSLPLGLPVLGAPPRLICDSRVLERYLLEAKEAENITTGCAEHCSLNENITVPDTKVNFYAWKRMEVGQQAVEVWQGLALLSEAVLRGQALLVNSSQPWEPLQLHVDKAVSGLRSLTTLLRALGAQKEAISPPDAASAAPLRTITADTFRKLFRVYSNFLRGKLKLYTGEACRTGDR

In an embodiment, the human EPO protein is the mature form of EPO (withthe signal peptide being cleaved off) which is a glycoprotein of 166amino acid residues (SEQ ID NO: 14) having the sequence of:

APPRLICDSRVLERYLLEAKEAENITTGCAEHCSLNENITVPDTKVNFYAWKRMEVGQQAVEVWQGLALLSEAVLRGQALLVNSSQPWEPLQLHVDKAVSGLRSLTTLLRALGAQKEAISPPDAASAAPLRTITADTFRKLFRVYSNFLR GKLKLYTGEACRTGDR

In various embodiments, the modified EPO protein is a mutant form of theEPO protein. In some embodiments, the mutations allow for the modifiedEPO protein to have one or more of attenuated activity, reducedendogenous activity, reduced binding affinity, and decreased specificbioactivity relative to unmutated, i.e. wild type EPO or other EPO-basedagents. For instance, one or more of attenuated activity, reducedendogenous activity, reduced binding affinity, and decreased specificbioactivity relative to unmutated, i.e. wild type EPO or other EPO-basedagents may be at an EPO receptor (EPOR) and/or an EphR. Consequentially,in various embodiments, the mutations allow for the modified EPO proteinto have reduced systemic toxicity, reduced side effects, and reducedoff-target effects relative to unmutated, i.e., wild type EPO protein orother EPO-based agents. In some embodiments, the present chimericproteins have a mutant EPO that has both mutations that attenuate EPObinding and/or activity at an EPOR and therefore allow for a morecontrolled, on-target therapeutic effect (e.g. relative wild type EPOprotein or other EPO-based agents).

In some embodiments, mutants that reduce, substantially reduce,substantially block, or block an EPO-EphR interaction are provided. Insome embodiments, there are provided mutations that reduce or inhibitEPO binding and/or activity at an EphR and therefore reduce tumorstimulating side effects (e.g. relative wild type EPO protein or otherEPO-based agents).

In some embodiments, the chimeric proteins have EPO mutants bearing amutation that affects interaction with an EPOR and EphR (e.g. viadouble, triple, etc., mutations). In some embodiments, the presentchimeric proteins have a mutant EPO that has both mutations thatattenuate EPO binding and/or activity at an EPOR and therefore allow fora more controlled, on-target therapeutic effect (e.g. relative wild typeEPO protein or other EPO-based agents) and mutations that reduce orinhibit EPO binding and/or activity at an EphR and therefore reducetumor stimulating side effects (e.g. relative wild type EPO protein orother EPO-based agents). These mutations may be at the same or atdifferent positions.

In various embodiments, the modified EPO protein comprises one or moremutations that attenuate the activity of the EPO protein. In variousembodiments, these mutations reduce or eliminate off-target activity ofthe EPO protein. In various embodiments, the modified EPO protein isactive on target cells because, despite reduced binding affinity (e.g.at an EPOR), the targeting moiety provides the missing binding affinityrequired for activation. In various embodiments, the modified EPOprotein is substantially inactive en route to the site of therapeuticactivity and has its effect substantially on specifically targeted celltypes which greatly reduces undesired side effects.

In various embodiments, the modified EPO protein comprises one or moremutations that reduce the endogenous activity of the EPO protein (e.g.at an EPOR) to about 75%, or about 70%, or about 60%, or about 50%, orabout 40%, or about 30%, or about 25%, or about 20%, or about 10%, orabout 5%, or about 3%, or about 1%, e.g., relative to wild type EPO orother EPO-based agents.

In various embodiments, the modified EPO protein comprises one or moremutations that cause the EPO protein to have reduced affinity, e.g.binding (e.g. K_(D)) or activation (e.g. EC₅₀), for one or morereceptors. In various embodiments, the modified EPO protein has about1%, or about 3%, about 5%, about 10%, about 15%, about 20%, about 25%,about 30%, about 35%, about 40%, about 45%, about 50%, about 60%, about65%, about 70%, about 75%, about 80%, about 85%, about 90%, about 95%,or about 10%-20%, about 20%-40%, about 50%, about 40%-60%, about60%-80%, about 80%-100% of the affinity for a receptor relative to wildtype EPO or other EPO-based agents. In some embodiments, the bindingaffinity is at least about 2-fold lower, about 3-fold lower, about4-fold lower, about 5-fold lower, about 6-fold lower, about 7-foldlower, about 8-fold lower, about 9-fold lower, at least about 10-foldlower, at least about 15-fold lower, at least about 20-fold lower, atleast about 25-fold lower, at least about 30-fold lower, at least about35-fold lower, at least about 40-fold lower, at least about 45-foldlower, at least about 50-fold lower, at least about 100-fold lower, atleast about 150-fold lower, or about 10-50-fold lower, about 50-100-foldlower, about 100-150-fold lower, about 150-200-fold lower, or more than200-fold lower relative to wild type EPO or other EPO-based agents. Insome embodiments, e.g. in comparing the mutant EPO binding to an EphR,the binding affinity may be effectively physiologically irrelevant.

In various embodiments, the modified EPO protein comprises one or moremutations that cause the EPO protein to have reduced affinity and/oractivity for an EPO receptor. Illustrative EPO receptors include, butare not limited to, an EPOR homodimer or an EPOR/CD131 heterodimer. Alsoincluded as an EPO receptor is beta-common receptor (βcR).

In various embodiments, the modified EPO protein comprises one or moremutations that cause the EPO protein to have reduced affinity for anephrin (Eph) receptor. Illustrative Eph receptors include, but are notlimited to, EPHA1, EPHA2, EPHA3, EPHA4, EPHA5, EPHA6, EPHA7, EPHA8,EPHA9, EPHA10, EPHB1, EPHB2, EPHB3, EPHB4, EPHB5, and EPHB6.

Various embodiments pertain to the modified EPO protein comprises one ormore mutations that cause the EPO protein to have attenuated activity atan EPOR that is restorable by the activity of the targeting moiety. Insome embodiments, this is in the context of a hematological cell (e.g.RBC, erythroid cell, etc.).

Various embodiments pertain to the modified EPO protein comprises one ormore mutations that cause the EPO protein to have severely reduced oreven ablated activity at an EPOR that is not substantially restorable bythe activity of the targeting moiety. For example, in some embodiments,this is in the context of a tumor cell.

Various embodiments pertain to the modified EPO protein comprises one ormore mutations that cause the EPO protein to have attenuated activity atan EphR that is restorable by the activity of the targeting moiety. Insome embodiments, this is in the context of a hematological cell (e.g.RBC, erythroid cell, etc.).

Various embodiments pertain to the modified EPO protein comprises one ormore mutations that cause the EPO protein to have severely reduced oreven ablated activity at an EphR that is not substantially restorable bythe activity of the targeting moiety. For example, in some embodiments,this is in the context of a tumor cell.

In some embodiments, the chimeric protein has a targeting moietydirecting to a hematological cell (e.g. RBC, erythroid cell, etc.) andhas a modified EPO that bears mutations which attenuate activity at anEPOR and also attenuate activity at an EphR. For example, and by way ofnon-limitation, such constructs would permit therapeutic effect in thecontext of a hematological cell having a hybrid EPOR/EphR receptor.

In some embodiments, the chimeric protein has a targeting moietydirecting to a hematological cell (e.g. RBC, erythroid cell, etc.) andhas a modified EPO that bears mutations which attenuate activity at anEPOR and substantially reduce or ablate activity at an EphR. Forexample, and by way of non-limitation, such constructs would permittherapeutic effect in the context of a hematological cell having a EPORwhile preventing en route binding of the EPO at an EphR receptor (and,for example, causing tumor stimulation).

Various embodiments pertain to the modified EPO protein comprising oneor more mutations that cause the EPO protein to have reduced affinityfor receptors that comprise one or more different EPO receptors or Ephreceptors (e.g. heterodimer, heterotrimers, etc., including by way ofnon-limitation: EPOR-EPHB4, EPOR-βcR-EPOR).

Also provided are the receptors of EP Patent Publication No. 2492355 theentire contents of which are hereby incorporated by reference, includingby way of non-limitation, NEPORs.

The structure of the human EPO protein is predicted to comprisefour-helix bundles including helices A, B, C, and D. In variousembodiments, the modified EPO protein comprises one or more mutationslocated in four regions of the EPO protein which are important forbioactivity, i.e., amino acid residues 10-20, 44-51, 96-108, and142-156. In some embodiments, the one or more mutations are located atresidues 11-15, 44-51, 100-108, and 147-151. These residues arelocalized to helix A (Val11, Arg14, and Tyr15), helix C (Ser100, Arg103,Ser104, and Leu108), helix D (Asn147, Arg150, Gly151, and Leu155), andthe NB connecting loop (residues 42-51). In some embodiments, themodified EPO protein comprises mutations in residues between amino acids41-52 and amino acids 147, 150, 151, and 155. Without wishing to bebound by theory, it is believed that mutations of these residues havesubstantial effects on both receptor binding and in vitro biologicalactivity. In some embodiments, the modified EPO protein comprisesmutations at residues 11, 14, 15, 100, 103, 104, and 108. Withoutwishing to be bound by theory, it is believed that mutations of theseresidues have modest effects on receptor binding activity and muchgreater effects on in vitro biological activity. Illustrativesubstitutions include, but are not limited to, one or more of Val11Ser,Arg14Ala, Arg14Gln, Tyr15Ile, Pro42Asn, Thr44Ile, Lys45Asp, Val46Ala,Tyr51Phe, Ser100Glu, Ser100Thr, Arg103Ala, Ser104Ile, Ser104Ala,Leu108Lys, Asn147Lys, Arg150Ala, Gly151Ala, and Leu155Ala.

In some embodiments, the chimeric proteins of the invention comprisemodified EPO protein having one or more mutations that cause the EPOprotein to have attenuated binding and/or activity at an EPOR. Exemplarymutations include amino acid substitutions including, but are notlimited to, one or more of V11S, R14A, R14E, R14Q, Y151, K20E, T441,K451, K45D, V46A, F48G, K97A, K97E, S100E, S100T, R103A, R103E, R103H,R103N, R103Q, S104A, S1041, L105A, L108A, L108K, R110E, R143A, N147K,R150A, R150Q, R150E, G151A, L155A, and L155N. Additional mutations thataffects the binding of EPO to EPOR include those disclosed in Syed R Set al. Nature (1998) 395(6701):511-6, Grodberg et al. Eur. J. Biochem.(1993) 218:597-601, and Elliott S, et al. (1997) Blood 89(2):493-502,the entire contents of all of which are hereby incorporated byreference.

In some embodiments, the chimeric proteins of the invention comprisemodified EPO protein having one or more mutations that cause the EPOprotein to have attenuated binding and/or activity at an EphR. Forexample, the chimeric proteins of the invention comprise modified EPOprotein having one or more mutations that cause the EPO protein to haveattenuated binding and/or activity at EphB4. In some embodiments, themodified EPO protein may include additional N-glycosylation and/orpegylation modifications at amino acids including, but not limited to,R14, K97, R103, Q115, 1119, E123, A128, R131, E55, E72, R76, K83, andS85. In other embodiments, the modified EPO protein may include one ormore modifications at the CD-loop (spanning amino acids G112-V136).Exemplary mutations in the CD-loop include mutations at amino acids119-128, e.g., mutating ISPPDAASAAPLRT (SEQ ID NO: 255) to GGPPGSGKGSPGG(SEQ ID NO: 256). Another exemplary mutation in the CD-loop includesmutations at amino acids 113-120, e.g., mutating GAQKEAIS (SEQ ID NO:257) to GGSGGSGG (SEQ ID NO: 258). In some embodiments, the modified EPOprotein may include one or more modifications in helix B (spanning aminoacids 58-82). For example, the modified EPO protein may include one ormore mutations at amino acids Q58, E62, Q65, L69, E72, R76, A79, andL80. Additional mutations that affects the binding of EPO to EphRinclude those disclosed in Pradeep et al. Cancer Cell (2015) 28:610-622,Boissel et al. (1993) JBC. 268(21):15983-15993, and Michael Brines etal. PNAS (2008), 105(31):10925-10930, the entire contents of all ofwhich are hereby incorporated by reference.

In some embodiments, the modified EPO protein comprises mutations thataffect bioactivity and not binding, e.g. those listed in Eliot, et al.Mapping of the Active Site of Recombinant Human Erythropoietin Jan. 15,1997; Blood: 89 (2), the entire contents of which are herebyincorporated by reference.

In some embodiments, the modified EPO protein comprises one or moremutations involving surface residues of the EPO protein which areinvolved in receptor contact. Without wishing to be bound by theory, itis believed that mutations of these surface residues are less likely toaffect protein folding thereby retaining some biological activity.Illustrative surface residues that may be mutated include, but are notlimited to, residues 147 and 150. In illustrative embodiments, themutations are substitutions including, one or more of N147A, N147K,R150A and R150E.

In some embodiments, the modified EPO protein comprises one or moremutations at residues N59, E62, L67, and L70, and one or more mutationsthat affect disulfide bond formation. Without wishing to be bound bytheory, it is believed that these mutations affect folding and/or arepredicted be in buried positions and thus affects biological activityindirectly.

In an embodiment, the modified EPO protein comprises a K20E substitutionwhich significantly reduces receptor binding. See Elliott, et al.,(1997) Blood, 89:493-502, the entire contents of which are herebyincorporated by reference.

Additional EPO mutations that may be incorporated into the chimeric EPOprotein of the invention are disclosed in, for example, Elliott, et al.,(1997) Blood, 89:493-502, and Taylor et al., (2010) PEDS, 23(4):251-260, the entire contents of all of which are hereby incorporated byreference.

In some embodiments, the modified EPO agent has one or more amino acidmutations disclosed herein with reference to SEQ ID NO:13 or 14. In anembodiment, the modified EPO agent has one or more amino acid mutationsdisclosed herein with reference to SEQ ID NO:14.

In some embodiments the modified EPO protein comprises one or moremutations that cause the EPO protein to have reduced affinity for areceptor that is lower than the binding affinity of the targeting moietyfor its receptor. In some embodiments, this binding affinitydifferential is between the modified EPO protein/receptor (e.g.EPO-EPOR) and targeting moiety/receptor on the same cell. In someembodiments, this binding affinity differential allows for the modifiedEPO protein to have localized, on-target effects and to minimizeoff-target effects that underlie side effects that are observed withwild type EPO proteins. In some embodiments, this binding affinity is atleast about 2-fold, or at least about 5-fold, or at least about 10-fold,or at least about 15-fold lower, or at least about 25-fold, or at leastabout 50-fold lower, or at least about 100-fold, or at least about150-fold less.

Illustrative EPO-based agents, which in some embodiments, are useful forcomparison to the present constructs include, but are not limited to,epoetin alfa, including without limitation, DARBEPOETIN (ARANESP),EPOCEPT (LUPIN PHARMA), NANOKINE (NANOGEN PHARMACEUTICAL), EPOFIT (INTASPHARMA), EPOGEN (AMGEN), EPOGIN, EPREX, (JANSSEN-CILAG), BINOCRIT(SANDOZ), PROCRIT; epoetin beta, including without limitation,NEORECORMON (HOFFMANN-LA ROCHE), RECORMON, Methoxy polyethyleneglycol-epoetin beta (MIRCERA, ROCHE); epoetin delta, including withoutlimitation, DYNEPO (erythropoiesis stimulating protein, SHIRE PLC);epoetin omega, including without limitation, EPOMAX; epoetin zeta,including without limitation, SILAPO (STADA) and RETACRIT (HOSPIRA) andother EPOs, including without limitation, EPOCEPT (LUPINPHARMACEUTICALS), EPOTRUST (PANACEA BIOTEC LTD), ERYPRO SAFE (BIOCONLTD.), REPOITIN (SERUM INSTITUTE OF INDIA LIMITED), VINTOR (EMCUREPHARMACEUTICALS), EPOFIT (INTAS PHARMA), ERYKINE (INTASBIOPHARMACEUTICA), WEPDX (WOCKHARDT BIOTECH), ESPOGEN (LG LIFESCIENCES), RELIPOIETIN (RELIANCE LIFE SCIENCES), SHANPOIETIN (SHANTHABIOTECHNICS LTD), ZYROP (CADILA HEALTHCARE LTD.), EPIAO (RHUEPO)(SHENYANG SUNSHINE PHARMACEUTICAL CO. LTD), CINNAPOIETIN (CINNAGEN).

In various embodiments, the signaling agent is a toxin or toxic enzyme.In some embodiments, the toxin or toxic enzyme is derived from plantsand bacteria. Illustrative toxins or toxic enzymes include, but are notlimited to, the diphtheria toxin, Pseudomonas toxin, anthrax toxin,ribosome-inactivating proteins (RIPs) such as ricin and saporin,modeccin, abrin, gelonin, and poke weed antiviral protein. Additionaltoxins include those disclosed in Mathew et al., (2009) Cancer Sci100(8): 1359-65, the entire disclosures are hereby incorporated byreference. In such embodiments, the chimeric proteins of the inventionmay be utilized to induce cell death in cell-type specific manner. Insuch embodiments, the toxin may be modified, e.g. mutated, to reduceaffinity and/or activity of the toxin for an attenuated effect, asdescribed with other signaling agents herein.

Targeting Moiety Cellular Recruitment

In various embodiments, the chimeric proteins of the present inventioncomprise a targeting moiety having antigen recognition domains whichspecifically bind to an antigen of interest. In various embodiments, theantigen may be found on any cells associated with a disease or disorder(e.g., cancer cells, immune cells such as those associated with allergicor chronic inflammatory disorders or reactions, infected cells, pathogencells, cells that are degenerating, or any other target cells ofinterest). In various embodiments, the antigen is found at the site ofdesired therapy, e.g. near the therapeutic receptor.

In various embodiments, the targeting moiety is directed to a target(e.g. antigen or epitope) which is present on the same cell as thetherapeutic receptor. In various embodiments, the targeting moiety isdirected to a target (e.g. antigen or epitope) which is not present onthe same cell as the other receptor.

In various embodiments, such antigens can be found on one or more tumorcells e.g. a tumor cell expressing one or more receptors. Tumor cells,or cancer cells refer to an uncontrolled growth of cells or tissuesand/or an abnormal increased in cell survival and/or inhibition ofapoptosis which interferes with the normal functioning of bodily organsand systems. For example, tumor cells include benign and malignantcancers, polyps, hyperplasia, as well as dormant tumors ormicrometastases. Illustrative tumor cells include, but are not limitedto cells of: basal cell carcinoma, biliary tract cancer; bladder cancer;bone cancer; brain and central nervous system cancer; breast cancer;cancer of the peritoneum; cervical cancer; choriocarcinoma; colon andrectum cancer; connective tissue cancer; cancer of the digestive system;endometrial cancer; esophageal cancer; eye cancer; cancer of the headand neck; gastric cancer (including gastrointestinal cancer);glioblastoma; hepatic carcinoma; hepatoma; intra-epithelial neoplasm;kidney or renal cancer; larynx cancer; leukemia; liver cancer; lungcancer (e.g., small-cell lung cancer, non-small cell lung cancer,adenocarcinoma of the lung, and squamous carcinoma of the lung);melanoma; myeloma; neuroblastoma; oral cavity cancer (lip, tongue,mouth, and pharynx); ovarian cancer; pancreatic cancer; prostate cancer;retinoblastoma; rhabdomyosarcoma; rectal cancer; cancer of therespiratory system; salivary gland carcinoma; sarcoma; skin cancer;squamous cell cancer; stomach cancer; testicular cancer; thyroid cancer;uterine or endometrial cancer; cancer of the urinary system; vulvalcancer; lymphoma including Hodgkin's and non-Hodgkin's lymphoma, as wellas B-cell lymphoma (including low grade/follicular non-Hodgkin'slymphoma (NHL); small lymphocytic (SL) NHL; intermediategrade/follicular NHL; intermediate grade diffuse NHL; high gradeimmunoblastic NHL; high grade lymphoblastic NHL; high grade smallnon-cleaved cell NHL; bulky disease NHL; mantle cell lymphoma;AIDS-related lymphoma; and Waldenstrom's Macroglobulinemia; chroniclymphocytic leukemia (CLL); acute lymphoblastic leukemia (ALL); Hairycell leukemia; chronic myeloblastic leukemia; as well as othercarcinomas and sarcomas; and post-transplant lymphoproliferativedisorder (PTLD), as well as abnormal vascular proliferation associatedwith phakomatoses, edema (e.g. that associated with brain tumors), andMeigs' syndrome.

Tumor cells, or cancer cells also include, but are not limited to,carcinomas, e.g. various subtypes, including, for example,adenocarcinoma, basal cell carcinoma, squamous cell carcinoma, andtransitional cell carcinoma), sarcomas (including, for example, bone andsoft tissue), leukemias (including, for example, acute myeloid, acutelymphoblastic, chronic myeloid, chronic lymphocytic, and hairy cell),lymphomas and myelomas (including, for example, Hodgkin and non-Hodgkinlymphomas, light chain, non-secretory, MGUS, and plasmacytomas), andcentral nervous system cancers (including, for example, brain (e.g.gliomas (e.g. astrocytoma, oligodendroglioma, and ependymoma),meningioma, pituitary adenoma, and neuromas, and spinal cord tumors(e.g. meningiomas and neurofibroma).

In various embodiments, the targeting moiety targets an antigen on acancer cell which allows the modified soluble agent to have atherapeutically relevant action on the cell via the therapeutic receptor(and, optionally, the modified soluble agent substantially avoidsinteraction with the other receptor).

Illustrative tumor antigens include, but are not limited to,MART-1/Melan-A, gp100, Dipeptidyl peptidase IV (DPPIV), adenosinedeaminase-binding protein (ADAbp), cyclophilin b, Colorectal associatedantigen (CRC)-0017-1A/GA733, Carcinoembryonic Antigen (CEA) and itsimmunogenic epitopes CAP-1 and CAP-2, etv6, aml1, Prostate SpecificAntigen (PSA) and its immunogenic epitopes PSA-1, PSA-2, and PSA-3,prostate-specific membrane antigen (PSMA), T-cell receptor/CD3-zetachain, MAGE-family of tumor antigens (e.g., MAGE-A1, MAGE-A2, MAGE-A3,MAGE-A4, MAGE-A5, MAGE-A6, MAGE-A7, MAGE-A8, MAGE-A9, MAGE-A10,MAGE-A11, MAGE-A12, MAGE-Xp2 (MAGE-B2), MAGE-Xp3 (MAGE-B3), MAGE-Xp4(MAGE-B4), MAGE-C1, MAGE-C2, MAGE-C3, MAGE-C4, MAGE-C5), GAGE-family oftumor antigens (e.g., GAGE-1, GAGE-2, GAGE-3, GAGE-4, GAGE-5, GAGE-6,GAGE-7, GAGE-8, GAGE-9), BAGE, RAGE, LAGE-1, NAG, GnT-V, MUM-1, CDK4,tyrosinase, p53, MUC family, HER2/neu, p21ras, RCAS1, α-fetoprotein,E-cadherin, α-catenin, β-catenin and γ-catenin, p120ctn, gp100 Pmel117,PRAME, NY-ESO-1, cdc27, adenomatous polyposis coli protein (APC),fodrin, Connexin 37, Ig-idiotype, p15, gp75, GM2 and GD2 gangliosides,viral products such as human papilloma virus proteins, Smad family oftumor antigens, Imp-1, NA, EBV-encoded nuclear antigen (EBNA)-1, brainglycogen phosphorylase, SSX-1, SSX-2 (HOM-MEL-40), SSX-1, SSX-4, SSX-5,SCP-1 CT-7, c-erbB-2, CD19, CD20, CD22, CD30, CD33, CD37, CD56, CD70,CD74, CD138, AGS16, MUC1, GPNMB, Ep-CAM, PD-L1, PD-L2, PMSA, and BCMA(TNFRSF17). In various embodiments, a targeting moiety binds one or moreof these tumor antigens.

In various embodiments, the present chimeric protein has a targetingmoiety directed against PD-1. In some embodiments, the chimeric proteinhas a targeting moiety which selectively binds a PD-1 polypeptide. Insome embodiments, the chimeric protein comprises one or more antibodies,antibody derivatives or formats, peptides or polypeptides, or fusionproteins that selectively bind a PD-1 polypeptide.

In an embodiment, the targeting moiety comprises the anti-PD-1 antibodypembrolizumab (aka MK-3475, KEYTRUDA), or fragments thereof.Pembrolizumab and other humanized anti-PD-1 antibodies are disclosed inHamid, et al. (2013) New England Journal of Medicine 369 (2): 134-44,U.S. Pat. No. 8,354,509, and WO 2009/114335, the entire disclosures ofwhich are hereby incorporated by reference. In illustrative embodiments,pembrolizumab or an antigen-binding fragment thereof for use in themethods provided herein comprises a heavy chain comprising the aminoacid sequence of:

SEQ ID NO: 15 QVQLVQSGVEVKKPGASVKVSCKASGYTFTNYYMYWVRQAPGQGLEWMGGINPSNGGTNFNEKFKNRVTLTTDSSTTTAYMELKSLQFDDTAVYYCARRDYRFDMGFDYWGQGTTVTVSSASTKGPSVFPLAPCSRSTSESTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTKTYTCNVDHKPSNTKVDKRVESKYGPPCPPCPAPEFLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSQEDPEVQFNWYVDGVEVHNAKTKPREEQFNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKGLPSSIEKTISKAKGQPREPQVYTLPPSQEEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSRLTVDKSRWQEGNVFSCSVMHEALHNHYTQKSLSLSLGK;and/or a light chain comprising the amino acid sequence of:

SEQ ID NO: 16 EIVLTQSPATLSLSPGERATLSCRASKGVSTSGYSYLHWYQQKPGQAPRLLIYLASYLESGVPARFSGSGSGTDFTLTISSLEPEDFAVYYCQHSRDLPLTFGGGTKVEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYA CEVTHQGLSSPVTKSFNRGEC.

In an embodiment, the targeting moiety comprises the anti-PD-1 antibody,nivolumab (aka BMS-936558, MDX-1106, ONO-4538, OPDIVO), or fragmentsthereof. Nivolumab (clone 5C4) and other human monoclonal antibodiesthat specifically bind to PD-1 are disclosed in U.S. Pat. No. 8,008,449and WO 2006/121168, the entire disclosures of which are herebyincorporated by reference. In illustrative embodiments, nivolumab or anantigen-binding fragment thereof comprises a heavy chain comprising theamino acid sequence of:

SEQ ID NO: 17 QVQLVESGGG VVQPGRSLRL DCKASGITFS NSGMHWVRQA PGKGLEWVAVIWYDGSKRYYADSVKGRFTI SRDNSKNTLF LQMNSLRAED TAVYYCATND DYWGQGTLVTVSSASTKGPSVFPLAPCSRS TSESTAALGC LVKDYFPEPV TVSWNSGALT SGVHTFPAVLQSSGLYSLSSVVTVPSSSLG TKTYTCNVDH KPSNTKVDKR VESKYGPPCP PCPAPEFLGGPSVFLFPPKPKDTLMISRTP EVTCVVVDVS QEDPEVQFNW YVDGVEVHNA KTKPREEQFNSTYRVVSVLTVLHQDWLNGK EYKCKVSNKG LPSSIEKTIS KAKGQPREPQ VYTLPPSQEEMTKNQVSLTCLVKGFYPSDI AVEWESNGQP ENNYKTTPPV LDSDGSFFLY SRLTVDKSRWQEGNVFSCSVMHEALHNHYT QKSLSLSLGK;and/or a light chain comprising the amino acid sequence of:

SEQ ID NO: 18 EIVLTQSPAT LSLSPGERAT LSCRASQSVS SYLAWYQQKP GQAPRLLIYDASNRATGIPARFSGSGSGTD FTLTISSLEP EDFAVYYCQQ SSNWPRTFGQ GTKVEIKRTVAAPSVFIFPPSDEQLKSGTA SVVCLLNNFY PREAKVQWKV DNALQSGNSQ ESVTEQDSKDSTYSLSSTLTLSKADYEKHK VYACEVTHQG LSSPVTKSFN RGEC.

In an embodiment, the targeting moiety comprises the anti-PD-1 antibodypidilizumab (aka CT-011, hBAT or hBAT-1), or fragments thereof.Pidilizumab and other humanized anti-PD-1 monoclonal antibodies aredisclosed in US 2008/0025980 and WO 2009/101611, the entire disclosuresof which are hereby incorporated by reference. In illustrativeembodiments, the anti-PD-1 antibody or an antigen-binding fragmentthereof for use in the methods provided herein comprises a light chainvariable regions comprising an amino acid sequence selected from SEQ IDNOS: 15-18 of US 2008/0025980:

SEQ ID No: 15 of US 2008/0025980 (SEQ ID NO: 19):EIVLTQSPSSLSASVGDRVTITCSARSSVSYMHWYQQKPGKAPKLLIYRTSNLASGVPSRFSGSGSGTDFTLTINSLQPEDFATYYCQQRSSFPLTFG GGTKLEIK; SEQ ID No:16 of US 2008/0025980 (SEQ ID NO: 20):EIVLTQSPSSLSASVGDRVTITCSARSSVSYMHWFQQKPGKAPKLWIYRTSNLASGVPSRFSGSGSGTDYTLTINSLQPEDFATYYCQQRSSFPLTFG GGTKLEIK; SEQ ID No:17 of US 2008/0025980 (SEQ ID NO: 21):EIVLTQSPSSLSASVGDRVTITCSARSSVSYMHWFQQKPGKAPKLWIYRTSNLASGVPSRFSGSGSGTDYCLTINSLQPEDFATYYCQQRSSFPLTFGG GTKLEIK; SEQ ID No:18 of US 2008/0025980 (SEQ ID NO: 22):EIVLTQSPSSLSASVGDRVTITCSARSSVSYMHWFQQKPGKAPKLWIYRTSNLASGVPSRFSGSGSGTSYCLTINSLQPEDFATYYCQQRSSFPLTFG GGTKLEIK;and/or a heavy chain comprising an amino acid sequence selected from SEQID NOS: 20-24 of US 2008/0025980:

SEQ ID No: 20 of US 2008/0025980 (SEQ ID NO: 23):QVQLVQSGSELKKPGASVKISCKASGYSFSNYGMNWVRQAPGQGLQWMGWINTDSGESTYAEEFKGRFVFSLDTSVSTAYLQITSLTAEDTGMYFCA KVGYDALDYWGQGTLVTVSS;SEQ ID No: 21 of US 2008/0025980 (SEQ ID NO: 24):QVQLVQSGSELKKPGASVKISCKASGYTFTNYGMNWVRQAPGQGLQWMGWINTDSGESTYAEEFKGRFVFSLDTSVSTAYLQITSLTAEDTGMYFCA KVGYDALDYWGQGTLVTVSS;SEQ ID No: 22 of US 2008/0025980 (SEQ ID NO: 25):QVQLVQSGSELKKPGASVKISCKASGYTFTNYGMNWVRQAPGQGLQWMGWINTDSGESTYAEEFKGRFVFSLDTSVNTAYLQITSLTAEDTGMYFCV RVGYDALDYWGQGTLVTVSS;SEQ ID No: 23 of US 2008/0025980 (SEQ ID NO: 26):QIQLVQSGSELKKPGASVKISCKASGYTFTNYGMNWVRQAPGQGLQWMGWINTDSGESTYAEEFKGRFVFSLDTSVNTAYLQITSLTAEDTGMYFCVR VGYDALDYWGQGTLVTVSS;SEQ ID No: 24 of US 2008/0025980 (SEQ ID NO: 27):QIQLVQSGSELKKPGASVKISCKASGYTFTNYGMNWVKQAPGQGLKWMGWINTDSGESTYAEEFKGRFAFSLDTSVNTAYLQITSLNAEDTGMYFCVR VGYDALDYWGQGTLVTVSS.

In an embodiment, the targeting moiety comprises a light chaincomprising SEQ ID NO:18 of US 2008/0025980 and a heavy chain comprisingSEQ ID NO:22 of US 2008/0025980.

In an embodiment, the targeting moiety comprises AMP-514 (akaMEDI-0680).

In an embodiment, the targeting moiety comprises the PD-L2-Fc fusionprotein AMP-224, which is disclosed in WO2010/027827 and WO 2011/066342,the entire disclosures of which are hereby incorporated by reference. Insuch an embodiment, the targeting moiety may include a targeting domainwhich comprises SEQ ID NO:4 of WO2010/027827 (SEQ ID NO: 28):

LFTVTVPKELYIIEHGSNVTLECNFDTGSHVNLGAITASLQKVENDTSPHRERATLLEEQLPLGKASFHIPQVQVRDEGQYQCIIIYGVAWDYKYLTLKVKASYRKINTHILKVPETDEVELTCQATGYPLAEVSWPNVSVPANTSHSRTPEGLYQVTSVLRLKPPPGRNFSCVFWNTHVRELTLASIDLQSQMEPRTHPTWLLHIFIPFCIIAFIFIATVIALRKQLCQKLYSSKDTTKRPVTTTKR EVNSAIand/or the B7-DC fusion protein which comprises SEQ ID NO:83 ofWO2010/027827 (SEQ ID NO: 29):

MIFLLLMLSLELQLHQIAALFTVTVPKELYIIEHGSNVTLECNFDTGSHVNLGAITASLQKVENDTSPHRERATLLEEQLPLGKASFHIPQVQVRDEGQYQCIIIYGVAWDYKYLTLKVKASYRKINTHILKVPETDEVELTCQATGYPLAEVSWPNVSVPANTSHSRTPEGLYQVTSVLRLKPPPGRNFSCVFWNTHVRELTLASIDLQSQMEPRTHPTWEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCWVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSL SLSPGK.

In an embodiment, the targeting moiety comprises the peptide AUNP 12 orany of the other peptides disclosed in US 2011/0318373 or 8,907,053. Forexample, the targeting moiety may comprise AUNP 12 (i.e., Compound 8 orSEQ ID NO:49 of US 2011/0318373) which has the sequence of:

In an embodiment, the targeting moiety comprises the anti-PD-1 antibody1E3, or fragments thereof, as disclosed in US 2014/0044738, the entiredisclosures of which are hereby incorporated by reference. Inillustrative embodiments, 1E3 or an antigen-binding fragment thereof foruse in the methods provided herein comprises a heavy chain variableregion comprising the amino acid sequence of:

SEQ ID NO: 31 EVQLQQSGPV LVKPGASVKM SCKASGYTFT DYYMNWVKQSHGKSLEWIGNINPYNGGTTY NQKFKGKATL TVDKSSRTAY MEINSLTSEDSAVYYCARGRIYDGSLDYWG QGTALTVSS;and/or a light chain variable region comprising the amino acid sequenceof:

SEQ ID NO: 32 DIQMTQFPSS LCASQGGKVT VTCKASQDIN NYMAWYQHKPGKGPRLLIHYTSTLLSGIPS RFSGSGSGRD YSFSISNLEP EDIATYYCLQ YDNLWTFGGGTKLEIK.

In an embodiment, the targeting moiety comprises the anti-PD-1 antibody1E8, or fragments thereof, as disclosed in US 2014/0044738, the entiredisclosures of which are hereby incorporated by reference. Inillustrative embodiments, 1E8 or an antigen-binding fragment thereof foruse in the methods provided herein comprises a heavy chain variableregion comprising the amino acid sequence of:

SEQ ID NO: 33 QVQLQQSGAE LAKPGASVRL SCKASGYTFT NYWMHWVKQR PGQGLEWIGHINPSSGFTTY NQNFKDKATL TADKSSNTAY MQLSSLTYED SAVYFCAREDYDVDYWGQGT TLTVSS;and/or a light chain variable region comprising the amino acid sequenceof:

SEQ ID NO: 34 DIVMTQSQKF MSTSVGDRVS VTCKASQSVD TNVAWYQQKPGQSPKALIFSASYRYSGVPD RFTGSGSGTD FTLTINSVQS EDLAEYFCQQ YNSYPYTFGSGTKLEIK.

In an embodiment, the targeting moiety comprises the anti-PD-1 antibody1H3, or fragments thereof, as disclosed in US 2014/0044738, the entiredisclosures of which are hereby incorporated by reference. Inillustrative embodiments, 1H3 or an antigen-binding fragment thereof foruse in the methods provided herein comprises a heavy chain variableregion comprising the amino acid sequence of:

SEQ ID NO: 35 EVQLVESGGG LVKPGGSLKL SCAASGFTFS DYGMHWVRQAPEKGLEWVAYISSGSYTIYY TDTVKGRFTI SRDNAKNTLF LQMTSLRSEDTAMYYCARRGYGSFYEYYFD YWGQGTTLTV SS;and/or light chain variable region comprising the amino acid sequenceof:

SEQ ID NO: 36 QIVLTQSPAL MSASPGEKVT MTCSASSSVS YMYWYQQKPRSSPKPWIYLTSNLASGVPAR FSGSGSGTSY SLTISSMEAE DAATYYCQQW SSNPFTFGSGTKLEIK.

In an embodiment, the targeting moiety comprises a nanobody directedagainst PD-1 as disclosed, for example, in U.S. Pat. No. 8,907,065 andWO 2008/071447, the entire disclosures of which are hereby incorporatedby reference. In illustrative embodiments, the nanobodies against PD-1comprise SEQ ID NOS: 347-351 of U.S. Pat. No. 8,907,065:

SEQ ID No: 347 of U.S. Pat. No. 8,907,065 (SEQ ID NO: 37):EVQLVESGGGLVQAGKSLRLSCAASGSIFSIHAMGWFRQAPGKEREFVAAITWSGGITYYEDSVKGRFTISRDNAKNTVYLQMNSLKPEDTAIYYCAA DRAESSWYDYWGQGTQVTVSS;SEQ ID No: 348 of U.S. Pat. No. 8,907,065 (SEQ ID NO: 38):EVQLVESGGGLVQAGGSLRLSCAASGSIASIHAMGWFRQAPGKEREFVAVITWSGGITYYADSVKGRFTISRDNAKNTVYLQMNSLKPEDTAIYYCAGDKHQSSWYDYWGQGTQVTVSS; SEQ ID No: 349 of U.S. Pat. No. 8,907,065 (SEQID NO: 39): EVQLVESGGGLVQAGGSLRLSCAASGSISSIHAMGWFRQAPGKEREFVAAITWSGGITYYADSLKGRFTISRDNAKNTGYLQMNSLKPEDTAIYYCAADRAQSSWYDYWGQGTQVTVSS; SEQ ID No: 350 of U.S. Pat. No. 8,907,065 (SEQID NO: 40): EVQLVESGGGLVQAGGSLGLSCAASGSIFSINAMAWFRQAPGKEREFVALISWSGGSTYYEDSVKGRFTISRDNAKNTVYLQMNSLKPEDTAIYYCA ADRVDSNWYDYWGQGTQVTVSS;SEQ ID No: 351 of U.S. Pat. No. 8,907,065 (SEQ ID NO: 41):EVQLVESGGGLVQAGGSLRLSCAASGRAFSSGTMGWFRRAPGKEREFVASIPWSGGRIYYADSVKGRFTISRDNAQNTVYLQMNSLKPEDTAVYYCAVKERSTGWDFASWGQCTQVTVSS.

In an embodiment, the targeting moiety comprises any one of theanti-PD-1 antibodies, or fragments thereof, as disclosed inUS2011/0271358 and WO2010/036959, the entire contents of which arehereby incorporated by reference. In illustrative embodiments, theantibody or an antigen-binding fragment thereof for use in the methodsprovided herein comprises a heavy chain comprising an amino acidsequence selected from SEQ ID NOS: 25-29 of US2011/0271358:

SEQ ID No: 25 of US2011/0271358 (SEQ ID NO: 42):QVQLVQSGAELKQPGASVKMSCKASGYSFTSSWIHWVKQAPGQGLEWIGYIYPSTGFTEYNQKFKDRATLTADKSTSTAYMELSSLRSEDSAVYYCARWRDSSGYHAMDYWGQGTSVTVSS; SEQ ID No: 26 of US2011/0271358 (SEQ ID NO:43): QVQLVQSGAEVKQPGASVKMSCKASGYSFTSSWIHWVKQAPGQGLEWIGYIYPSTGFTEYNQKFKDRATLTADKSTSTAYMELSSLRSEDTAVYY3/d10CARWRDSSGYHAMDYWGQGTSVTVSS; SEQ ID No: 27 of US2011/0271358 (SEQ IDNO: 44): QVQLVQSGHEVKQPGASVKMSCKASGYSFTSSWIHWVKQAPGQGLEWIGYIYPSTGFTEYNQKFKDRATLTADKSTSTAYMELSSLRSEDTAVYYCARWRDSSGYHAMDYWGQGTLVTVSS; SEQ ID No: 28 of US2011/0271358 (SEQ ID NO:45): QVQLVQSGHEVKQPGASVKMSCKASGYSFTSSWIHWVRQAPGQGLEWIGYIYPSTGFTEYNQKFKDRATLTADKSTSTAYMELSSLRSEDTAVYYCARWRDSSGYHAMDYWGQGTLVTVSS; SEQ ID No: 29 of US2011/0271358 (SEQ ID NO:46): QVQLVQSGHEVKQPGASVKVSCKASGYSFTSSWIHWVRQAPGQGLEWIGYIYPSTGFTEYNQKFKDRATITADKSTSTAYMELSSLRSEDTAVYYCARWRDSSGYHAMDYWGQGTLVTVSS;and/or a light chain comprising an amino acid sequence selected from SEQID NOS: 30-33 of US2011/0271358:

SEQ ID No: 30 of US2011/0271358 (SEQ ID NO: 47):DIVLTQSPASLTLSPGQRLTISCRASQSVSTSGYSYMHWYQQKPDQSPKLLIKFGSNLESGIPARFSGSGSGTDFTLTISSLEEEDFATYYCQHSWEI PYTFGQGTKLEIK; SEQ IDNo: 31 of US2011/0271358 (SEQ ID NO: 48):DIVLTQSPATLSLSPGQRLTISCRASQSVSTSGYSYMHWYQQKPDQSPKLLIKFGSNLESGIPARFSGSGSGTDFTLTISSLEPEDFATYYCQHSWEIP YTFGQGTKLEIK; SEQ IDNo: 32 of US2011/0271358 (SEQ ID NO: 49):EIVLTQSPATLSLSPGQRLTISCRASQSVSTSGYSYMHWYQQKPDQSPKLLIKFGSNLESGIPARFSGSGSGTDFTLTISSLEPEDFATYYCQHSWEIP YTFGQGTKLEIK; SEQ IDNo: 33 of US2011/0271358 (SEQ ID NO: 50):DIVLTQSPATLSLSPGQRLTISCRASQSVSTSGYSYMHWYQQKPDQSPKLLIKFGSNLESGIPARFSGSGSGTDFTLTISSLEPEDFAVYYCQHSWEIP YTFGQGTKLEIK.

In various embodiments, the present chimeric protein comprises one ormore antibodies directed against PD-1, or antibody fragments thereof,selected from TSR-042 (Tesaro, Inc.), REGN2810 (RegeneronPharmaceuticals, Inc.), PDR001 (Novartis Pharmaceuticals), and BGB-A317(BeiGene Ltd.)

In various embodiments, the present chimeric protein has a targetingmoiety directed against PD-L1. In some embodiments, the chimeric proteinhas a targeting moiety which selectively bind a PD-L1 polypeptide. Insome embodiments, the chimeric protein comprises one or more antibodies,antibody derivatives or formats, peptides or polypeptides, or fusionproteins that selectively bind a PD-L1 polypeptide.

In an embodiment, the targeting moiety comprises the anti-PD-L1 antibodyMEDI4736 (aka durvalumab), or fragments thereof. MEDI4736 is selectivefor PD-L1 and blocks the binding of PD-L1 to the PD-1 and CD80receptors. MEDI4736 and antigen-binding fragments thereof for use in themethods provided herein comprises a heavy chain and a light chain or aheavy chain variable region and a light chain variable region. Thesequence of MEDI4736 is disclosed in WO/2016/06272, the entire contentsof which are hereby incorporated by reference. In illustrativeembodiments, MEDI4736 or an antigen-binding fragment thereof for use inthe methods provided herein comprises a heavy chain comprising the aminoacid sequence of:

SEQ ID NO: 51 EVQLVESGGG LVQPGGSLRL SCAASGFTFS RYWMSWVRQA PGKGLEWVANIKQDGSEKYY VDSVKGRFTI SRDNAKNSLY LQMNSLRAED TAVYYCAREG GWFGELAFDYWGQGTLVTVS SASTKGPSVF PLAPSSKSTS GGTAALGCLV KDYFPEPVTV SWNSGALTSGVHTFPAVLQS SGLYSLSSVV TVPSSSLGTQ TYICNVNHKP SNTKVDKRVE PKSCDKTHTCPPCPAPEFEG GPSVFLFPPK PKDTLMISRT PEVTCVVVDV SHEDPEVKFN WYVDGVEVHNAKTKPREEQY NSTYRVVSVL TVLHQDWLNG KEYKCKVSNK ALPASIEKTI SKAKGQPREPQVYTLPPSRE EMTKNQVSLT CLVKGFYPSD IAVEWESNGQ PENNYKTTPP VLDSDGSFFLYSKLTVDKSR WQQGNVFSCS VMHEALHNHY TQKSLSLSPG K;and/or a light chain comprising the amino acid sequence of:

SEQ ID NO: 52 EIVLTQSPGT LSLSPGERAT LSCRASQRVS SSYLAWYQQK PGQAPRLLIYDASSRATGIP DRFSGSGSGT DFTLTISRLE PEDFAVYYCQQYGSLPWTFGQGTKVEIKRTVAAPSVFIFP PSDEQLKSGT ASVVCLLNNF YPREAKVQWKVDNALQSGNS QESVTEQDSKDSTYSLSSTL TLSKADYEKH KVYACEVTHQGLSSPVTKSF NRGEC.

In illustrative embodiments, the MEDI4736 or an antigen-binding fragmentthereof for use in the methods provided herein comprises a heavy chainvariable region comprising the amino acid sequence of SEQ ID NO:4 ofWO/2016/06272

(SEQ ID NO: 53): EVQLVESGGGLVQPGGSLRLSCAASGFTFSRYWMSWVRQAPGKGLEWVANIKQDGSEKYYVDSVKGRFTISRDNAKNSLYLQMNSLRAEDTAVYYCAREGGWFGELAFDYWGQGTLVTVSS;and/or a light chain variable region comprising the amino acid sequenceof SEQ ID NO:3 of WO/2016/06272

(SEQ ID NO: 54): EIVLTQSPGTLSLSPGERATLSCRASQRVSSSYLAWYQQKPGQAPRLLIYDASSRATGIPDRFSGSGSGTDFTLTISRLEPEDFAVYYCQQYGSLPWT FGQGTKVEIK

In an embodiment, the targeting moiety comprises the anti-PD-L1 antibodyatezolizumab (aka MPDL3280A, RG7446, Tecentriq), or fragments thereof.In illustrative embodiments, atezolizumab or an antigen-binding fragmentthereof for use in the methods provided herein comprises a heavy chaincomprising the amino acid sequence of:

SEQ ID NO: 55 EVQLVESGGGLVQPGGSLRLSCAASGFTFSDSWIHWVRQAPGKGLEWVAWISPYGGSTYYADSVKGRFTISADTSKNTAYLQMNSLRAEDTAVYYCARRHWPGGFDYWGQGTLVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYASTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK;and/or a light chain comprising the amino acid sequence of:

SEQ ID NO: 56 DIQMTQSPSSLSASVGDRVTITCRASQDVSTAVAWYQQKPGKAPKLLIYSASFLYSGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQYLYHPATFGQGTKVEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEV THQGLSSPVTKSFNRGEC.

In an embodiment, the targeting moiety comprises the anti-PD-L1 antibodyavelumab (aka MSB0010718C), or fragments thereof. In illustrativeembodiments, avelumab or an antigen-binding fragment thereof for use inthe methods provided herein comprises a heavy chain comprising the aminoacid sequence of:

SEQ ID NO: 57 EVQLLESGGG LVQPGGSLRL SCAASGFTFS SYIMMWVRQAPGKGLEWVSSIYPSGGITFY ADTVKGRFTI SRDNSKNTLY LQMNSLRAEDTAVYYCARIKLGTVTTVDYW GQGTLVTVSS ASTKGPSVFP LAPSSKSTSGGTAALGCLVKDYFPEPVTVS WNSGALTSGV HTFPAVLQSS GLYSLSSVVTVPSSSLGTQTYICNVNHKPS NTKVDKKVEP KSCDKTHTCP PCPAPELLGGPSVFLFPPKPKDTLMISRTP EVTCVWDVS HEDPEVKFNW YVDGVEVHNAKTKPREEQYNSTYRVVSVLT VLHQDWLNGK EYKCKVSNKA LPAPIEKTISKAKGQPREPQVYTLPPSRDE LTKNQVSLTC LVKGFYPSDI AVEWESNGQPENNYKTTPPVLDSDGSFFLY SKLTVDKSRW QQGNVFSCSV MHEALHNHYT QKSLSLSPGK;and/or a light chain comprising the amino acid sequence of:

SEQ ID NO: 58 QSALTQPASV SGSPGQSITI SCTGTSSDVG GYNYVSWYQQ HPGKAPKLMIYDVSNRPSGV SNRFSGSKSG NTASLTISGL QAEDEADYYC SSYTSSSTRV FGTGTKVTVLGQPKANPTVT LFPPSSEELQ ANKATLVCLI SDFYPGAVTV AWKADGSPVK AGVETTKPSKQSNNKYAASS YLSLTPEQWK SHRSYSCQVT HEGSTVEKTV APTECS.

In an embodiment, the targeting moiety comprises the anti-PD-L1 antibodyBMS-936559 (aka 12A4, MDX-1105), or fragments thereof, as disclosed inUS 2013/0309250 and WO2007/005874, the entire disclosures of which arehereby incorporated by reference. In illustrative embodiments,BMS-936559 or an antigen-binding fragment thereof for use in the methodsprovided herein comprises a heavy chain variable region comprising theamino acid sequence of:

SEQ ID NO: 59 QVQLVQSGAEVKKPGSSVKVSCKTSGDTFSTYAISWVRQAPGQGLEWMGGIIPIFGKAHYAQKFQGRVTITADESTSTAYMELSSLRSEDTAVYFCARKFHFVSGSPFGMDVWGQGTTVTVSS;and/or a light chain variable region comprising the amino acid sequenceof:

SEQ ID NO: 60 EIVLTQSPATLSLSPGERATLSCRASQSVSSYLAWYQQKPGQAPRLLIYDASNRATGIPARFSGSGSGTDFTLTISSLEPEDFAVYYCQQRSNWPTFG QGTKVEIK.

In an embodiment, the targeting moiety comprises the anti-PD-L1 antibody3G10, or fragments thereof, as disclosed in US 2013/0309250 andWO2007/005874, the entire disclosures of which are hereby incorporatedby reference. In illustrative embodiments, 3G10 or an antigen-bindingfragment thereof for use in the methods provided herein comprises aheavy chain variable region comprising the amino acid sequence of:

SEQ ID NO: 61 QVQLVQSGAEVKKPGASVKVSCKASGYTFTDYGFSWVRQAPGQGLEWMGWITAYNGNTNYAQKLQGRVTMTTDTSTSTVYMELRSLRSDDTAVYYCAR DYFYGMDVWGQGTTVTVSS;and/or a light chain variable region comprising the amino acid sequenceof:

SEQ ID NO: 62 EIVLTQSPATLSLSPGERATLSCRASQSVSSYLVWYQQKPGQAPRLLIYDASNRATGIPARFSGSGSGTDFTLTISSLEPEDFAVYYCQQRSNWPRTF GQGTKVEIK.

In an embodiment, the targeting moiety comprises the anti-PD-L1 antibody10A5, or fragments thereof, as disclosed in US 2013/0309250 andWO2007/005874, the entire disclosures of which are hereby incorporatedby reference. In illustrative embodiments, 10A5 or an antigen-bindingfragment thereof for use in the methods provided herein comprises aheavy chain variable region comprising the amino acid sequence of:

SEQ ID NO: 63 QVQLVQSGAEVKKPGASVKVSCKASGYTFTSYDVHWVRQAPGQRLEWMGWLHADTGITKFSQKFQGRVTITRDTSASTAYMELSSLRSEDTAVYYCA RERIQLWFDYWGQGTLVTVSS;and/or a light chain variable region comprising the amino acid sequenceof:

SEQ ID NO: 64 DIQMTQSPSSLSASVGDRVTITCRASQGISSWLAWYQQKPEKAPKSLIYAASSLQSGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQYNSYPYTFGQ GTKLEIK.

In an embodiment, the targeting moiety comprises the anti-PD-L1 antibody5F8, or fragments thereof, as disclosed in US 2013/0309250 andWO2007/005874, the entire disclosures of which are hereby incorporatedby reference. In illustrative embodiments, 5F8 or an antigen-bindingfragment thereof for use in the methods provided herein comprises aheavy chain variable region comprising the amino acid sequence of:

SEQ ID NO: 65 QVQLVQSGAEVKKPGSSVKVSCKVSGGIFSTYAINWVRQAPGQGLEWMGGIIPIFGTANHAQKFQGRVTITADESTSTAYMELSSLRSEDTAVYYCARDQ GIAAALFDYWGQGTLVTVSS;and/or a light chain variable region comprising the amino acid sequenceof:

SEQ ID NO: 66 EIVLTQSPGTLSLSPGERATLSCRASQSVSSSYLAWYQQKPGQAPRLLIYGASSRATGIPDRFSGSGSGTDFTLTISRLEPEDFAVYYCQQYGSSPWTFG QGTKVEIK

In an embodiment, the targeting moiety comprises the anti-PD-L1 antibody10H10, or fragments thereof, as disclosed in US 2013/0309250 andWO2007/005874, the entire disclosures of which are hereby incorporatedby reference. In illustrative embodiments, 10H10 or an antigen-bindingfragment thereof for use in the methods provided herein comprises aheavy chain variable region comprising the amino acid sequence of:

SEQ ID NO: 67 EVQLVESGGGLVQPGRSLRLSCAVSGFTFDDYVVHWVRQAPGKGLEWVSGISGNSGNIGYADSVKGRFTISRDNAKNSLYLQMNSLRAEDTALYYCAVPF DYWGQGTLVTVSS;and/or a light chain variable region comprising the amino acid sequenceof:

SEQ ID NO: 68 DIQMTQSPSSLSASVGDRVTITCRASQGISSWLAWYQQKPEKAPKSLIYAASSLQSGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQYNSYPYTFGQ GTKLEIK.

In an embodiment, the targeting moiety comprises the anti-PD-L1 antibody1B12, or fragments thereof, as disclosed in US 2013/0309250 andWO2007/005874, the entire disclosures of which are hereby incorporatedby reference. In illustrative embodiments, 1B12 or an antigen-bindingfragment thereof for use in the methods provided herein comprises aheavy chain variable region comprising the amino acid sequence of:

SEQ ID NO: 69 QVQLVQSGAEVKKPGSSVKVSCKTSGDTFSSYAISWVRQAPGQGLEWMGGIIPIFGRAHYAQKFQGRVTITADESTSTAYMELSSLRSEDTAVYFCARKFHFVSGSPFGMDVWGQGTTVTVSS;and/or a light chain variable region comprising the amino acid sequenceof:

SEQ ID NO: 70 EIVLTQSPATLSLSPGERATLSCRASQSVSSYLAWYQQKPGQAPRLLIYDASNRATGIPARFSGSGSGTDFTLTISSLEPEDFAVYYCQQRSNWPTFGQG TKVEIK.

In an embodiment, the targeting moiety comprises the anti-PD-L1 antibody7H1, or fragments thereof, as disclosed in US 2013/0309250 andWO2007/005874, the entire disclosures of which are hereby incorporatedby reference. In illustrative embodiments, 7H1 or an antigen-bindingfragment thereof for use in the methods provided herein comprises aheavy chain variable region comprising the amino acid sequence of:

SEQ ID NO: 71 QVQLVQSGAEVKKPGSSVKVSCKTSGGTFSSYAISWVRQAPGQGLEWMGGIIPIFGKAHYAQKFQGRVTITADESTTTAYMELSSLRSEDTAVYYCARKYDYVSGSPFGMDVWGQGTTVTVSS;and/or a light chain variable region comprising the amino acid sequenceof:

SEQ ID NO: 72 EIVLTQSPATLSLSPGERATLSCRASQSVSSYLAWYQQKPGQAPRLLIYDASNRATGIPARFSGSGSGTDFTLTISSLEPEDFAVYYCQQRSNWPTFGQG TKVEIK.

In an embodiment, the targeting moiety comprises the anti-PD-L1 antibody11E6, or fragments thereof, as disclosed in US 2013/0309250 andWO2007/005874, the entire disclosures of which are hereby incorporatedby reference. In illustrative embodiments, 11E6 or an antigen-bindingfragment thereof for use in the methods provided herein comprises aheavy chain variable region comprising the amino acid sequence of:

SEQ ID NO: 73 QVQLVQSGAEVKKPGSSVKVSCKASGGTFSSYAINWVRQAPGQGLEWMGGIIPIFGSANYAQKFQDRVTITADESTSAAYMELSSLRSEDTAVYYCARDSSGWSRYYMDVWGQGTTVTVSS;and/or a light chain variable region comprising the amino acid sequenceof:

SEQ ID NO: 74 EIVLTQSPGTLSLSPGERATLSCRASQSVSSSYLAWYQQKPGQAPRLLIYGASSRATGIPDRFSGSGSGTDFTLTISRLEPEDFAVYYCQQYGSSPFGGG TKVEIK.

In an embodiment, the targeting moiety comprises the anti-PD-L1 antibody12B7, or fragments thereof, as disclosed in US 2013/0309250 andWO2007/005874, the entire disclosures of which are hereby incorporatedby reference. In illustrative embodiments, 12B7 or an antigen-bindingfragment thereof for use in the methods provided herein comprises aheavy chain variable region comprising the amino acid sequence of:

SEQ ID NO: 75 QVQLVQSGAEVKEPGSSVKVSCKASGGTFNSYAISWVRQAPGQGLEWMGGIIPLFGIAHYAQKFQGRVTITADESTNTAYMDLSSLRSEDTAVYYCARKYSYVSGSPFGMDVWGQGTTVTVSS;and/or a light chain variable region comprising the amino acid sequenceof:

SEQ ID NO: 76 EIVLTQSPATLSLSPGERATLSCRASQSVSSYLAWYQQKPGQAPRLLIYDASNRATGIPARFSGSGSGTDFTLTISSLEPEDFAVYYCQQRSNWPTFGQG TRLEIK.

In an embodiment, the targeting moiety comprises the anti-PD-L1 antibody13G4, or fragments thereof, as disclosed in US 2013/0309250 andWO2007/005874, the entire disclosures of which are hereby incorporatedby reference. In illustrative embodiments, 13G4 or an antigen-bindingfragment thereof for use in the methods provided herein comprises aheavy chain variable region comprising the amino acid sequence of:

SEQ ID NO: 77 EVQLVESGGGLVQPGRSLRLSCAASGITFDDYGMHWVRQAPGKGLEWVSGISWNRGRIEYADSVKGRFTISRDNAKNSLYLQMNSLRAEDTALYYCAKGRFRYFDWFLDYWGQGTLVTVSS;and/or a light chain variable region comprising the amino acid sequenceof:

SEQ ID NO: 78 AIQLTQSPSSLSASVGDRVTITCRASQGISSALAWYQQKPGKAPKLLIYDASSLESGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQFNSYPFTFGP GTKVDIK.

In an embodiment, the targeting moiety comprises the anti-PD-L1 antibody1E12, or fragments thereof, as disclosed in US 2014/0044738, the entiredisclosures of which are hereby incorporated by reference. Inillustrative embodiments, 1E12 or an antigen-binding fragment thereoffor use in the methods provided herein comprises a heavy chain variableregion comprising the amino acid sequence of:

SEQ ID NO: 79 EVKLQESGPS LVKPSQTLSL TCSVTGYSIT SDYWNWIRKFPGNKLEYVGYISYTGSTYYN PSLKSRISIT RDTSKNQYYL QLNSVTSEDTATYYCARYGGWLSPFDYWGQ GTTLTVSS;and/or a light chain variable region comprising the amino acid sequenceof:

SEQ ID NO: 80 DIVMTQSHKL MSTSVGDRVS ITCKASQDVG TAVAWYQQKPGQSPKLLIYWASTRHTGVPD RFTGSGSGTD FTLTISNVQS EDLADYFCQQ DSSYPLTFGAGTKVELK.

In an embodiment, the targeting moiety comprises the anti-PD-L1 antibody1F4, or fragments thereof, as disclosed in US 2014/0044738, the entiredisclosures of which are hereby incorporated by reference. Inillustrative embodiments, 1F4 or an antigen-binding fragment thereof foruse in the methods provided herein comprises a heavy chain variableregion comprising the amino acid sequence of:

SEQ ID NO: 81 EVQLQESGPG LVAPSQSLSI TCTVSGFSLT TYSINWIRQPPGKGLEWLGVMWAGGGTNSN SVLKSRLIIS KDNSKSQVFL KMNSLQTDDTARYYCARYYGNSPYYAIDYW GQGTSVTVSS;and/or a light chain variable region comprising the amino acid sequenceof:

SEQ ID NO: 82 DIVTTQSHKL MSTSVGDRVS ITCKASQDVG TAVAWYQQKPGQSPKLLIYWASTRHTGVPD RFTGSGSGTD FTLTISNVQS EDLADYFCQQ DSSYPLTFGAGTKVELK.

In an embodiment, the targeting moiety comprises the anti-PD-L1 antibody2G11, or fragments thereof, as disclosed in US 2014/0044738, the entiredisclosures of which are hereby incorporated by reference. Inillustrative embodiments, 2G11 or an antigen-binding fragment thereoffor use in the methods provided herein comprises a heavy chain variableregion comprising the amino acid sequence of:

SEQ ID NO: 83 EVKLQESGPS LVKPSQTLSL TCSVTGYSII SDYWNWIRKFPGNKLEYLGYISYTGSTYYN PSLKSRISIT RDTSKNQYYL QLNSVTTEDTATYYCARRGGWLLPFDYWGQ GTTLTVSS;and/or a light chain variable region comprising the amino acid sequenceof:

SEQ ID NO: 84 DIVMTQSPSS LAVSVGEKVS MGCKSSQSLL YSSNQKNSLAWYQQKPGQSPKLLIDWASTR ESGVPDRFTG SGSGTDFTLT ISSVKAEDLAVYYCQQYYGYPLTFGAGTKL ELK.

In an embodiment, the targeting moiety comprises the anti-PD-L1 antibody3B6, or fragments thereof, as disclosed in US 2014/0044738, the entiredisclosures of which are hereby incorporated by reference. Inillustrative embodiments, 3B6 or an antigen-binding fragment thereof foruse in the methods provided herein comprises a heavy chain variableregion comprising the amino acid sequence of:

SEQ ID NO: 85 EVKLQESGPS LVKPGASVKL SCKASGYTFT SYDINWVKQRPGQGLEWIGWIFPRDNNTKY NENFKGKATL TVDTSSTTAY MELHSLTSEDSAVYFCTKENWVGDFDYWGQ GTTLTLSS;and/or a light chain variable region comprising the amino acid sequenceof:

SEQ ID NO: 86 DIVMTQSPAI MSASPGEKVT MTCSASSSIR YMHWYQQKPGTSPKRWISDTSKLTSGVPAR FSGSGSGTSY ALTISSMEAE DAATYYCHQR SSYPWTFGGGTKLEIK.

In an embodiment, the targeting moiety comprises the anti-PD-L1 antibody3D10, or fragments thereof, as disclosed in US 2014/0044738 andWO2012/145493, the entire disclosures of which are hereby incorporatedby reference. In illustrative embodiments, 3D10 or an antigen-bindingfragment thereof for use in the methods provided herein comprises aheavy chain variable region comprising the amino acid sequence of:

SEQ ID NO: 87 EVQLQQSGPD LVTPGASVRI SCQASGYTFP DYYMNWVKQSHGKSLEWIGDIDPNYGGTTY NQKFKGKAIL TVDRSSSTAY MELRSLTSEDSAVYYCARGALTDWGQGTSL TVSS;and/or a light chain variable region comprising the amino acid sequenceof:

SEQ ID NO: 88 QIVLSQSPAI LSASPGEKVT MTCRASSSVS YIYWFQQKPGSSPKPWIYATFNLASGVPAR FSGSGSGTSY SLTISRVETE DAATYYCQQW SNNPLTFGAGTKLELK.

In an embodiment, the targeting moiety comprises any one of theanti-PD-L1 antibodies disclosed in US2011/0271358 and WO2010/036959, theentire contents of which are hereby incorporated by reference. Inillustrative embodiments, the antibody or an antigen-binding fragmentthereof for use in the methods provided herein comprises a heavy chaincomprising an amino acid sequence selected from SEQ ID Nos: 34-38 ofUS2011/0271358:

SEQ ID No: 34 of US2011/0271358 (SEQ ID NO: 89):EVQLVQSGPELKKPGASVKMSCKASGYTFTSYVMHWVKQAPGQRLEWIGYVNPFNDGTKYNEMFKGRATLTSDKSTSTAYMELSSLRSEDSAVYYCARQ AWGYPWGQGTLVTVSS; SEQID No: 35 of US2011/0271358 (SEQ ID NO: 90):EVQLVQSGAEVKKPGASVKMSCKASGYTFTSYVMHWVKQAPGQRLEWIGYVNPFNDGTKYNEMFKGRATLTSDKSTSTAYMELSSLRSEDTAVYYCARQ AWGYPWGQGTLVTVSS; SEQID No: 36 of US2011/0271358 (SEQ ID NO: 91):EVQLVQSGAEVKKPGASVKMSCKASGYTFTSYVMHWVRQAPGQRLEWIGYVNPFNDGTKYNEMFKGRATLTSDKSTSTAYMELSSLRSEDTAVYYCARQ AWGYPWGQGTLVTVSS; SEQID No: 37 of US2011/0271358 (SEQ ID NO: 92):EVQLVQSGAEVKKPGASVKVSCKASGYTFTSYVMHWVRQAPGQRLEWIGYVNPFNDGTKYNEMFKGRATLTSDKSTSTAYMELSSLRSEDTAVYYCARQ AWGYPWGQGTLVTVSS; SEQID No: 38 of US2011/0271358 (SEQ ID NO: 93):EVQLVQSGAEVKKPGASVKVSCKASGYTFTSYVMHWVRQAPGQRLEWIGYVNPFNDGTKYNEMFKGRATITSDKSTSTAYMELSSLRSEDTAVYYCARQ AWGYPWGQGTLVTVSS;and/or a light chain comprising an amino acid sequence selected from SEQID Nos: 39-42 of US2011/0271358:

SEQ ID No: 39 of US2011/0271358 (SEQ ID NO: 94):DIVLTQSPASLALSPGERATLSCRATESVEYYGTSLVQWYQQKPGQPPKLLIYAASSVDSGVPSRFSGSGSGTDFTLTINSLEEEDAAMYFCQQSRRV PYTFGQGTKLEIK; SEQ IDNo: 40 of US2011/0271358 (SEQ ID NO: 95):DIVLTQSPATLSLSPGERATLSCRATESVEYYGTSLVQWYQQKPGQPPKLLIYAASSVDSGVPSRFSGSGSGTDFTLTINSLEAEDAAMYFCQQSRRV PYTFGQGTKLEIK; SEQ IDNo: 41 of US2011/0271358 (SEQ ID NO: 96):EIVLTQSPATLSLSPGERATLSCRATESVEYYGTSLVQWYQQKPGQPPKLLIYAASSVDSGVPSRFSGSGSGTDFTLTINSLEAEDAAMYFCQQSRRV PYTFGQGTKLEIK; SEQ IDNo: 42 of US2011/0271358 (SEQ ID NO: 97):DIVLTQSPATLSLSPGERATLSCRATESVEYYGTSLVQWYQQKPGQPPKLLIYAASSVDSGVPSRFSGSGSGTDFTLTINSLEAEDAATYFCQQSRRV PYTFGQGTKLEIK.

In an embodiment, the targeting moiety comprises the anti-PD-L1 antibody2.7A4, or fragments thereof, as disclosed in WO 2011/066389, U.S. Pat.No. 8,779,108, and US2014/0356353, the entire disclosures of which arehereby incorporated by reference. In illustrative embodiments, 2.7A4 oran antigen-binding fragment thereof for use in the methods providedherein comprises a heavy chain variable region comprising the amino acidsequence of:

SEQ ID No: 2 of WO 2011/066389 (SEQ ID NO: 98):EVQLVESGGGLVKPGGSLRLSCAASGFTFSTYSMNWVRQAPGKGLEWVSSISSSGDYIYYADSVKGRFTISRDNAKNSLFLQMNSLKAEDTAVYYCARDLVTSMVAFDYWGQGTLVTVSS;and/or a light chain variable region comprising the amino acid sequenceof:

SEQ ID No: 7 of WO 2011/066389 (SEQ ID NO: 99):SYELTQPPSVSVSPGQAARITCSGDALPQKYVFWYQQKSGQAPVLVIYEDSKRPSGIPERFSGSSSGTMATLTISGAQVEDEADYYCYSTDRSGNHRVFG GGTRLTVL.

In an embodiment, the targeting moiety comprises the anti-PD-L1 antibody2.9D10, or fragments thereof, as disclosed in WO 2011/066389, U.S. Pat.No. 8,779,108, and US2014/0356353, the entire disclosures of which arehereby incorporated by reference. In illustrative embodiments, 2.9D10 oran antigen-binding fragment thereof for use in the methods providedherein comprises a heavy chain variable region comprising the amino acidsequence of:

SEQ ID No: 12 of WO 2011/066389 (SEQ ID NO: 100):EVQLVESGGGLVQPGGSLRLSCAASGFTFSSYWMSWVRQAPGKGLEWVANIKQDGGEQYYVDSVKGRFTISRDNAKNSLYLQMNSLRAEDTAVYYCARDWNYGYYDMDVWGQGTTVTVSS;and/or a light chain variable region comprising the amino acid sequenceof:

SEQ ID No: 17 of WO 2011/066389 (SEQ ID NO: 101):EIVLTQSPGTLSLSPGERATLSCRASQSVSSNYLAWFQQKPGQAPRLLIFGTSSRATGIPDRFSGSGSGTDFTLTISRLEPEDFAVYYCQQYGSSIFTFG PGTKVDIK.

In an embodiment, the targeting moiety comprises the anti-PD-L1 antibody2.14H9, or fragments thereof, as disclosed in WO 2011/066389, U.S. Pat.No. 8,779,108, and US2014/0356353, the entire disclosures of which arehereby incorporated by reference. In illustrative embodiments, 2.14H9 oran antigen-binding fragment thereof for use in the methods providedherein comprises a heavy chain variable region comprising the amino acidsequence of:

SEQ ID No: 22 of WO 2011/066389 (SEQ ID NO: 102):EVQLVESGGGLVQPGGSLRLSCAASGFTFSRYWMSWVRQAPGKGLEWVANIKQDGSEKYYVDSVKGRFTISRDNAKNSLYLQMNSLRAEDTAVYYCAREGGWFGELAFDYWGQGTLVTVSS;and/or a light chain variable region comprising the amino acid sequenceof:

SEQ ID No: 27 of WO 2011/066389 (SEQ ID NO: 103):EIVLTQSPGTLSLSPGERATLSCRASQRVSSSYLAWYQQKPGQAPRLLIYDASSRATGIPDRFSGSGSGTDFTLTISRLEPEDFAVYYCQQYGSLPWTFG QGTEVEIK

In an embodiment, the targeting moiety comprises the anti-PD-L1 antibody2.20A8, or fragments thereof, as disclosed in WO 2011/066389, U.S. Pat.No. 8,779,108, and US2014/0356353, the entire disclosures of which arehereby incorporated by reference. In illustrative embodiments, 2.20A8 oran antigen-binding fragment thereof for use in the methods providedherein comprises a heavy chain variable region comprising the amino acidsequence of:

SEQ ID No: 32 of WO 2011/066389 (SEQ ID NO: 104):EVQLLESGGGLVQPGGSLRLSCAASGFTFSNYAMSWVRQAPGKGLEWVSAIRGSGGSTYYADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCAKDLHYDSSGYLDYWGQGTLVTVSS;and/or a light chain variable region comprising the amino acid sequenceof:

SEQ ID No: 37 of WO 2011/066389 (SEQ ID NO: 105):DIQMTQSPSSVSASVGDRVTITCRASQGIRSWLAWYQQKPGKAPKLLIYAISRLQSGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQANSFPLTFGG GTKVEIK.

In an embodiment, the targeting moiety comprises the anti-PD-L1 antibody3.15G8, or fragments thereof, as disclosed in WO 2011/066389, U.S. Pat.No. 8,779,108, and US2014/0356353, the entire disclosures of which arehereby incorporated by reference. In illustrative embodiments, 3.15G8 oran antigen-binding fragment thereof for use in the methods providedherein comprises a heavy chain variable region comprising the amino acidsequence of:

SEQ ID No: 42 of WO 2011/066389 (SEQ ID NO: 106):EVQLVESGGGLVQPGGSLRLSCAASGFTFSSYWMSWVRQAPGKGLEWVANIKQDGGEKYYVDSVKGRFTISRDNAKNSLFLQMNSLRAEDTAVYYCARVQLYSDYFDYWGQGTLVTVSS;and/or a light chain variable region comprising the amino acid sequenceof:

SEQ ID No: 47 of WO 2011/066389 (SEQ ID NO: 107):DIQMTQSPSSVSASVGDRVTITCRASQGISSWLAWYQQKSGKAPKLLIYAASGLQSGVPSRFSGSGSGTDFTLTISSLQPEDLATYYCQQSHSLPPTFGQ GTKVEIK.

In an embodiment, the targeting moiety comprises the anti-PD-L1 antibody3.18G1, or fragments thereof, as disclosed in WO 2011/066389, U.S. Pat.No. 8,779,108, and US2014/0356353, the entire disclosures of which arehereby incorporated by reference. In illustrative embodiments, 3.18G1 oran antigen-binding fragment thereof for use in the methods providedherein comprises a heavy chain variable region comprising the amino acidsequence of:

SEQ ID No: 52 of WO 2011/066389 (SEQ ID NO: 108):EVQLLESGGDLVQPGGSLRLSCAASGFTFNSYAMSWVRQAPGKGLEWVSTISGSGGFTFSADSVKGRFTISRDNSKNTLFLQMNSLRVEDSAVYSCAKVLVGFNNGCWDYWGQGTLVTVSS:and/or a light chain variable region comprising the amino acid sequenceof:

SEQ ID No: 57 of WO 2011/066389 (SEQ ID NO: 109):SYVLTQPPSVSVAPGQTARITCGGNNIGSKSVHWYQQKPGQAPVLVVYDDSDRPSGIPERFSGSNSGNTATLTISRVEAGDEADYYCQVWDSSNDHVVFG GGTKLTVL.

In an embodiment, the targeting moiety comprises the anti-PD-L1 antibody2.7A4OPT, or fragments thereof, as disclosed in WO 2011/066389, U.S.Pat. No. 8,779,108, and US2014/0356353, and US2014/0356353, the entiredisclosures of which are hereby incorporated by reference. Inillustrative embodiments, 2.7A4OPT or an antigen-binding fragmentthereof for use in the methods provided herein comprises a heavy chainvariable region comprising the amino acid sequence of:

SEQ ID No: 62 of WO 2011/066389 (SEQ ID NO: 110):EVQLVESGGGLVKPGGSLRLSCAASGFTFSTYSMNWVRQAPGKGLEWVSSISSSGDYIYYADSVKGRFTISRDNAKNSLYLQMNSLRAEDTAVYYCARDLVTSMVAFDYWGQGTLVTVSS;and/or a light chain variable region comprising the amino acid sequenceof:

SEQ ID No: 67 of WO 2011/066389 (SEQ ID NO: 111):SYELTQPPSVSVSPGQTARITCSGDALPQKYVFWYQQKSGQAPVLVIYEDSKRPSGIPERFSGSSSGTMATLTISGAQVEDEADYYCYSTDRSGNHRVFG GGTKLTVL.

In an embodiment, the targeting moiety comprises the anti-PD-L1 antibody2.14H9OPT, or fragments thereof, as disclosed in WO 2011/066389, U.S.Pat. No. 8,779,108, and US2014/0356353, the entire disclosures of whichare hereby incorporated by reference. In illustrative embodiments,2.14H9OPT or an antigen-binding fragment thereof for use in the methodsprovided herein comprises a heavy chain variable region comprising theamino acid sequence of:

SEQ ID No: 72 of WO 2011/066389 (SEQ ID NO: 112):EVQLVESGGGLVQPGGSLRLSCAASGFTFSRYWMSWVRQAPGKGLEWVANIKQDGSEKYYVDSVKGRFTISRDNAKNSLYLQMNSLRAEDTAVYYCAREGGWFGELAFDYWGQGTLVTVSS;and/or a light chain variable region comprising the amino acid sequenceof:

SEQ ID No: 77 of WO 2011/066389 (SEQ ID NO: 113):EIVLTQSPGTLSLSPGERATLSCRASQRVSSSYLAWYQQKPGQAPRLLIYDASSRATGIPDRFSGSGSGTDFTLTISRLEPEDFAVYYCQQYGSLPWTFG QGTKVEIK

In an embodiment, the targeting moiety comprises any one of theanti-PD-L1 antibodies disclosed in WO2016/061142, the entire contents ofwhich are hereby incorporated by reference. In illustrative embodiments,the antibody or an antigen-binding fragment thereof for use in themethods provided herein comprises a heavy chain comprising an amino acidsequence selected from SEQ ID Nos: 18, 30, 38, 46, 50, 54, 62, 70, and78 of WO2016/061142:

SEQ ID No: 18 of WO2016/061142 (SEQ ID NO: 114):QVQLVQSGAEVKKPGASVKVSCKASGYTFTSYWMYWVRQATGQGLEWMGRIDPNSGSTKYNEKFKNRFTISRDDSKNTAYLQMNSLKTEDTAVYYCARDYRKGLYAMDYWGQGTTVTVSS; SEQ ID No: 30 of WO2016/061142 (SEQ ID NO: 115):EVQLVQSGAEVKKPGATVKISCKVSGYTFTSYWMYWVRQATGQGLEWMGRIDPNSGSTKYNEKFKNRVTITADKSTSTAYMELSSLRSEDTAVYYCARDYRKGLYAMDYWGQGTTVTVSS; SEQ ID No: 38 of WO2016/061142 (SEQ ID NO: 116):EVQLVQSGAEVKKPGESLRISCKGSGYTFTSYWMYWVRQAPGQGLEWMGRIDPNSGSTKYNEKFKNRVTISVDTSKNQFSLKLSSVTAADTAVYYCARDYRKGLYAMDYWGQGTTVTVSS; SEQ ID No: 46 of WO2016/061142 (SEQ ID NO: 117):EVQLVQSGAEVKKPGATVKISCKVSGYTFTSYWMYWIRQSPSRGLEWLGRIDPNSGSTKYNEKFKNRLTISKDTSKNQVVLTMTNMDPVDTATYYCARDYRKGLYAMDYWGQGTTVTVSS; SEQ ID No: 50 of WO2016/061142 (SEQ ID NO: 118):EVQLVQSGAEVKKPGESLRISCKGSGYTFTSYWMYWIRQPPGKGLEWIGRIDPNSGSTKYNEKFKNRVTITADKSTSTAYMELSSLRSEDTAVYYCARDY RKGLYAMDYWGQGTTVTVSS;SEQ ID No: 54 of WO2016/061142 (SEQ ID NO: 119):QVQLVQSGAEVKKPGASVKVSCKASGYTFTSYWMYWIRQSPSRGLEWLGRIDPNSGSTKYNEKFKNRFTISRDDSKNTAYLQMNSLKTEDTAVYYCARDYRKGLYAMDYWGQGTTVTVSS; SEQ ID No: 62 of WO2016/061142 (SEQ ID NO: 120):EVQLVQSGAEVKKPGESLRISCKGSGYTFTSYWMYWVRQARGQRLEWIGRIDPNSGSTKYNEKFKNRLTISKDTSKNQVVLTMTNMDPVDTATYYCARDYRKGLYAMDYWGQGTTVTVSS; SEQ ID No: 70 of WO2016/061142 (SEQ ID NO: 121):QITLKESGPTLVKPTQTLTLTCTFSGYTFTSYWMYWVRQAPGKGLEWVSRIDPNSGSTKYNEKFKNRVTITADKSTSTAYMELSSLRSEDTAVYYCARDY RKGLYAMDYWGQGTTVTVSS;SEQ ID No: 78 of WO2016/061142 (SEQ ID NO: 122):EVQLVQSGAEVKKPGATVKISCKVSGYTFTSYWMYWVRQARGQRLEWIGRIDPNSGSTKYNEKFKNRFTISRDNSKNTLYLQMNSLRAEDTAVYYCARDYRKGLYAMDYWGQGTTVTVSS;and/or a light chain comprising an amino acid sequence selected from SEQID Nos: 22, 26, 34, 42, 58, 66, 74, 82, and 86 of WO2016/061142:

SEQ ID No: 22 of WO2016/061142 (SEQ ID NO: 123):DIVMTQTPLSLPVTPGEPASISCKASQDVGTAVAWYLQKPGQSPQLLIYWASTRHTGIPARFSGSGSGTEFTLTISSLQSEDFAVYYCQQYNSYPLTFGQ GTKVEIK; SEQ ID No:26 of WO2016/061142 (SEQ ID NO: 124):DIQMTQSPSSLSASVGDRVTITCKASQDVGTAVAWYLQKPGQSPQLLIYWASTRHTGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQYNSYPLTFGQ GTKVEIK; SEQ ID No:34 of WO2016/061142 (SEQ ID NO: 125):EIVLTQSPDFQSVTPKEKVTITCKASQDVGTAVAWYLQKPGQSPQLLIYW ASTRHTGVPDRFSGSGSGTDFTLKISRVEAEDVGVYYCQQYNSYPLTFGQGTKVEIK; SEQ ID No: 42 ofWO2016/061142 (SEQ ID NO: 126):EIVLTQSPDFQSVTPKEKVTITCKASQDVGTAVAWYLQKPGQSPQLLIYWASTRHTGVPSRFSGSGSGTDFTFTISSLQPEDIATYYCQQYNSYPLTFGQ GTKVEIK. SEQ ID No:58 of WO2016/061142 (SEQ ID NO: 127):EIVLTQSPATLSLSPGERATLSCKASQDVGTAVAWYLQKPGQSPQLLIYWASTRHTGIPPRFSGSGYGTDFTLTINNIESEDAAYYFCQQYNSYPLTFGQ GTKVEIK; SEQ ID No:66 of WO2016/061142 (SEQ ID NO: 128):DVVMTQSPLSLPVTLGQPASISCKASQDVGTAVAWYQQKPGQAPRLLIYWASTRHTGVPSRFSGSGSGTEFTLTISSLQPDDFATYYCQQYNSYPLTFGQ GTKVEIK; SEQ ID No:74 of WO2016/061142 (SEQ ID NO: 129):DIQMTQSPSSLSASVGDRVTITCKASQDVGTAVAWYQQKPGQAPRLLIYWASTRHTGVPSRFSGSGSGTDFTFTISSLQPEDIATYYCQQYNSYPLTFGQ GTKVEIK; SEQ ID No:82 of WO2016/061142 (SEQ ID NO: 130):AIQLTQSPSSLSASVGDRVTITCKASQDVGTAVAWYLQKPGQSPQLLIYWASTRHTGVPSRFSGSGSGTDFTFTISSLEAEDAATYYCQQYNSYPLTFG QGTKVEIK; SEQ ID No:86 of WO2016/061142 (SEQ ID NO: 131):EIVLTQSPDFQSVTPKEKVTITCKASQDVGTAVAWYQQKPGQAPRLLIYWASTRHTGVPSRFSGSGSGTEFTLTISSLQPDDFATYYCQQYNSYPLTFG QGTKVEIK.

In an embodiment, the targeting moiety comprises any one of theanti-PD-L1 antibodies disclosed in WO2016/022630, the entire contents ofwhich are hereby incorporated by reference. In illustrative embodiments,the antibody or an antigen-binding fragment thereof for use in themethods provided herein comprises a heavy chain comprising an amino acidsequence selected from SEQ ID Nos: 2, 6, 10, 14, 18, 22, 26, 30, 34, 38,42, and 46 of WO2016/022630:

SEQ ID No: 2 of WO2016/022630 (SEQ ID NO: 132):EVKLVESGGGLVKPGGSLKLSCAASGFIFRSYGMSWVRQTPEKRLEWVASISSGGSTYYPDSVKGRFTISRDNARNILYLQMSSLRSEDTAMYDCARGYD SGFAYWGQGTLVTVSE; SEQID No: 6 of WO2016/022630 (SEQ ID NO: 133):EVKLVESGGGLVKPGGSLKLSCAASGFTFRSYGMSWVRQTPEKRLEWVASISSGGTTYYPDSVKGRFIISRDNARNILYLQMSSLRSEDTAMYYCAKGYD SGFAYWGQGTLVIVSA; SEQID No: 10 of WO2016/022630 (SEQ ID NO: 134):QVQLKQSGPGLVQPSQSLSITCTVSGFSLTTYGVHWVRQSPGKGLEWLGVIWRGVTTDYNAAFMSRLTITKDNSKSQVFFKMNSLQANDTAIYYCARLGF YAMDYWGQGTSVTVSS; SEQID No: 14 of WO2016/022630 (SEQ ID NO: 135):QVQLKQSGPGLVQPSQSLSITCTVSGFSLTSYGVHWVRQSPGKGLEWLGVIWSGGVTDYNAAFISRLSISKDNSKSQVFFKMNSLQANDTAIYYCARLGF YAMDYWGQGTSVTVSS; SEQID No: 18 of WO2016/022630 (SEQ ID NO: 136):EVKLFESGGGLVQPGGSLKLSCVASGFDFSTYWMHWVRQAPGQGLEWIGQINPDSTTINYAPSLKDRFIISRDNAKNTLFLQMSKVRSEDTALYYCAKPG DYGYDFDCWGQGTTLTVSS;SEQ ID No: 22 of WO2016/022630 (SEQ ID NO: 137):EVQLQESGPSLVKPSQTLSLTCSVTGDSITSGYWNWIRKFPGNKLEYMGYISYSGSTYYNPSLKSRISITRDTSKNQYYLQLNSVTTEDTATYYCARSLL WFSTGFAYWGQGTLVTVSA;SEQ ID No: 26 of WO2016/022630 (SEQ ID NO: 138):QVQLKQSGPGLVQPSQSLSITCTVSGFSLTSYGVHWVRQSPGKGLEWLGVIWSGGITDYNAAFKSRLSISKDNSKSQVFFKMNSLQANDTAIYFCARLGF YAMDYWGQGTSVTVSS; SEQID No: 30 of WO2016/022630 (SEQ ID NO: 139):EVKLVESGGGLVKPGGSLKLSCAASGFTFRSYGMSWARQIPEKRLEWVASISSGGTTYYLGSVQGRFTISRDNARNILYLQMSSLRSEDTAMYYCARGYD AGFAYWGQGTLVSVSE; SEQID No: 34 of WO2016/022630 (SEQ ID NO: 140):EVQLQESGPSLVKPSQTLSLTCSVTGDSITSGYWTWIRKFPGNKLEYMGYISYTGSTYYNPSLKSRISISRDTSKSQYYLQLNSVTTEDTATYYCARQRD WLGFAYWGQGTLVTVSA;SEQ ID No: 38 of WO2016/022630 (SEQ ID NO: 141):EEKLVESGGGLVKPGGSLKLSCAASGFSFSSYGMSWVRQTPEKRLEWVASISSGGSIYYPDSVKGRFTISRDNARNILYLQMSSLRSEDTAMYYCARGYD AGFAFWGQGTLVTASA; SEQID No: 42 of WO2016/022630 (SEQ ID NO: 142):QITLKESGPTLVKPTQTLTLTCTVSGFSLSTYGVHWIRQPPGKALEWLGVIWRGVTTDYNAAFMSRLTITKDNSKNQWLTMNNMDPVDTATYYCARLGFY AMDYWGQGTLVTVSS; SEQID No: 46 of WO2016/022630 (SEQ ID NO: 143):EVQLVESGGGLVKPGGSLRLSCAASGFIFRSYGMSWVRQAPGKGLEWVASISSGGSTYYPDSVKGRFTISRDNAKNSLYLQMNSLRAEDTAVYDCARGYD SGFAYWGQGTLVTVSS;and/or a light chain comprising an amino acid sequence selected from SEQID Nos: 4, 8, 12, 16, 20, 24, 28, 32, 36, 40, 44, and 48 ofWO2016/022630:

SEQ ID No: 4 of WO2016/022630 (SEQ ID NO: 144):DIVLTQSPASLAVSLGQRATISCRASQSVSTSSSSFMHWYQQKPGQPPKLLIKYASNLESGVPARFSGSGSGTDFTLNIHPVEEEDTATYYCQHSWEIPY TFGGGTKLEIKR; SEQ IDNo: 8 of WO2016/022630 (SEQ ID NO: 145):DIVLTQSPPSLAVSLGQRATISCRASQSVSTSSSSYMHWYQQKPGQPPKLLIKYASNLESGVPARFSGSGSGTDFTLNIHPVEEEDTATYYCQHSWEIPY TFGGGTKLEIK; SEQ IDNo: 12 of WO2016/022630 (SEQ ID NO: 146):SIVMTQTPKFLLVSAGDRVTITCKASQSVSNDVAWYQQKPGQSPKLLIYYAANRYTGVPDRFTGSGYGTDFTFTISIVQAEDLAVYFCQQDYTSPYTFGG GTKLEIK; SEQ ID No:16 of WO2016/022630 (SEQ ID NO: 147):SIVMTQTPKFLLVSAGDRVTITCKASQSVSNDVGWYQQKPGQSPKLLIYYASNRYSGVPDRFTGSGYGTDFTFTISTVQAEDLAVYFCQQDYTSPYTFGG GTKLEIK; SEQ ID No:20 of WO2016/022630 (SEQ ID NO: 148):DVLMTQTPLYLPVSLGDQASISCRSSQIIVHSNANTYLEWFLQKPGQSPKLLIYKVSNRFSGVPDRFSGSGSGTDFTLKISRVEAEDLGVYYCFQGSHVP YTFGGGTKLEIK; SEQ IDNo: 24 of WO2016/022630 (SEQ ID NO: 149):QIVLTQSPAIMSASPGEKVTLTCSASSSVSSSYLYWNQQKPGSSPKVWIYNTSNLASGVPARFSGSGSGTSYSLTISSMEAEDAASYFCHQWRSYPPTLG AGTKLELK; SEQ ID No:28 of WO2016/022630 (SEQ ID NO: 150):QIVLTQSPAIMSASPGEKVTMTCSANSSVSYMHWYQQKSGTSPKRWIYDTSKLASGVPARFSGSGSGTSYSLTISSMGAEDAATYYCQQWSSNPWTFGGG TKLEIK; SEQ ID No: 32of WO2016/022630 (SEQ ID NO: 151):DIVLTQSPASLAVSLGQRATISCRASQSVSTSSYSYMHWYQQKPGQPPKLLIKYASNLESGVPARFSGSGSGTDFTLNIHPVEEEDTATYYCQNSWEIPY TFGGGTKLEIK; SEQ IDNo: 36 of WO2016/022630 (SEQ ID NO: 152):DIVMTQTPSSLAVSLGEKVTMSCKSSQSLLYSSNQKNSLAWYQQKPGQSPKLLIYWASNRESGVPDRFTGSSSGTDFTLTISSVKAEDLAVYYCQQYYSY PLTFGAGTKLELK; SEQ IDNo: 40 of WO2016/022630 (SEQ ID NO: 153):DIVLTQSPASLAVSLGQRATISCRASQSVSTSSYSYVHWYQQKPGQPPKLLIKYASNLESGVPARFSGSGSGTDFTLNIHPVEEEDTATYYCQHSWEIPY TFGGGTKLEIK; SEQ IDNo: 44 of WO2016/022630 (SEQ ID NO: 154):DIQMTQSPSSLSASVGDRVTITCKASQSVSNDVAWYQQKPGKAPKLLIYYAANRYTGVPDRFSGSGYGTDFTFTISSLQPEDIATYFCQQDYTSPYTFGQ GTKLEIK; SEQ ID No:48 of WO2016/022630 (SEQ ID NO: 155):DIVLTQSPASLAVSPGQRATITCRASQSVSTSSSSFMHWYQQKPGQPPKLLIKYASNLESGVPARFSGSGSGTDFTLTINPVEANDTANYYCQHSWEIPY TFGQGTKLEIK.

In an embodiment, the targeting moiety comprises any one of theanti-PD-L1 antibodies disclosed in WO2015/112900, the entire contents ofwhich are hereby incorporated by reference. In illustrative embodiments,the antibody or an antigen-binding fragment thereof for use in themethods provided herein comprises a heavy chain comprising an amino acidsequence selected from SEQ ID Nos: 38, 50, 82, and 86 of WO 2015/112900:

SEQ ID No: 38 of WO2015/112900 (SEQ ID NO: 156):EVQLVQSGAEVKKPGESLRISCKGSGYTFTTYWMHWVRQATGQGLEWMGNIYPGTGGSNFDEKFKNRVTITADKSTSTAYMELSSLRSEDTAVYYCTRWT TGTGAYWGQGTTVTVSS;SEQ ID No: 50 of WO 2015/112900 (SEQ ID NO: 157):EVQLVQSGAEVKKPGESLRISCKGSGYTFTTYWMHWIRQSPSRGLEWLGNIYPGTGGSNFDEKFKNRFTISRDNSKNTLYLQMNSLRAEDTAVYYCTRWT TGTGAYWGQGTTVTVSS;SEQ ID No: 82 of WO 2015/112900 (SEQ ID NO: 158):QVQLVQSGAEVKKPGASVKVSCKASGYTFTTYWMHWIRQSPSRGLEWLGNIYPGTGGSNFDEKFKNRFTISRDNSKNTLYLQMNSLRAEDTAVYYCTRWT TGTGAYWGQGTTVTVSS;SEQ ID No: 86 of WO 2015/112900 (SEQ ID NO: 159):EVQLVQSGAEVKKPGESLRISCKGSGYTFTTYWMHWVRQAPGQGLEWMGNIYPGTGGSNFDEKFKNRFTISRDNSKNTLYLQMNSLRAEDTAVYYCTRWT TGTGAYWGQGTTVTVSS;and/or a light chain comprising an amino acid sequence selected from SEQID Nos: 42, 46, 54, 58, 62, 66, 70, 74, and 78 of WO 2015/112900:

SEQ ID No: 42 of WO2015/112900 (SEQ ID NO: 160):EIVLTQSPATLSLSPGERATLSCKSSQSLLDSGNQKNFLTWYQQKPGQAPRLLIYWASTRESGVPSRFSGSGSGTEFTLTISSLQPDDFATYYCQNDYSY PYTFGQGTKVEIK; SEQ IDNo: 46 of WO 2015/112900 (SEQ ID NO: 161):DIQMTQSPSSLSASVGDRVTITCKSSQSLLDSGNQKNFLTWYQQKPGQAPRLLIYWASTRESGIPPRFSGSGYGTDFTLTINNIESEDAAYYFCQNDYSY PYTFGQGTKVEIK; SEQ IDNo: 54 of WO 2015/112900 (SEQ ID NO: 162):EIVLTQSPATLSLSPGERATLSCKSSQSLLDSGNQKNFLTWYQQKPGKAPKLLIYWASTRESGVPSRFSGSGSGTDFTFTISSLQPEDIATYYCQNDYSY PYTFGQGTKVEIK; SEQ IDNo: 58 of WO 2015/112900 (SEQ ID NO: 163):DIVMTQTPLSLPVTPGEPASISCKSSQSLLDSGNQKNFLTWYQQKPGQAPRLLIYWASTRESGVPSRFSGSGSGTDFTFTISSLEAEDAATYYCQNDYSY PYTFGQGTKVEIK; SEQ IDNo: 62 of WO 2015/112900 (SEQ ID NO: 164):EIVLTQSPATLSLSPGERATLSCKSSQSLLDSGNQKNFLTWYQQKPGKAPKLLIYWASTRESGVPSRFSGSGSGTDFTFTISSLEAEDAATYYCQNDYSY PYTFGQGTKVEIK; SEQ IDNo: 66 of WO 2015/112900 (SEQ ID NO: 165):EIVLTQSPDFQSVTPKEKVTITCKSSQSLLDSGNQKNFLTWYQQKPGQAPRLLIYWASTRESGVPSRFSGSGSGTDFTFTISSLEAEDAATYYCQNDYSY PYTFGQGTKVEIK; SEQ IDNo: 70 of WO 2015/112900 (SEQ ID NO: 166):EIVLTQSPATLSLSPGERATLSCKSSQSLLDSGNQKNFLTWYQQKPGQAPRLLIYWASTRESGVPSRFSGSGSGTDFTFTISSLEAEDAATYYCQNDYSY PYTFGQGTKVEIK; SEQ IDNo: 74 of WO 2015/112900 (SEQ ID NO: 167):DIQMTQSPSSLSASVGDRVTITCKSSQSLLDSGNQKNFLTWYLQKPGQSPQLLIYWASTRESGVPSRFSGSGSGTDFTFTISSLEAEDAATYYCQNDYSY PYTFGQGTKVEIK; SEQ IDNo: 78 of WO 2015/112900 (SEQ ID NO: 168):DVVMTQSPLSLPVTLGQPASISCKSSQSLLDSGNQKNFLTWYQQKPGKAPKLLIYWASTRESGVPSRFSGSGSGTDFTFTISSLEAEDAATYYCQNDYSY PYTFGQGTKVEIK.

In an embodiment, the targeting moiety comprises any one of theanti-PD-L1 antibodies disclosed in WO 2010/077634 and U.S. Pat. No.8,217,149, the entire disclosures of which are hereby incorporated byreference. In illustrative embodiments, the anti-PD-L1 antibody or anantigen-binding fragment thereof for use in the methods provided hereincomprises a heavy chain region comprising the amino acid sequence of:

SEQ ID No: 20 of WO 2010/077634 (SEQ ID NO: 169):EVQLVESGGGLVQPGGSLRLSCAASGFTFSDSWIHWVRQAPGKGLEWVAWISPYGGSTYYADSVKGRFTISADTSKNTAYLQMNSLRAEDTAVYYCARRH WPGGFDYWGQGTLVTVSA;and/or a light chain variable region comprising the amino acid sequenceof:

SEQ ID No: 21 of WO 2010/077634 (SEQ ID NO: 170):DIQMTQSPSSLSASVGDRVTITCRASQDVSTAVAWYQQKPGKAPKLLIYSASFLYSGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQYLYHPATFGQ GTKVEIKR

In an embodiment, the targeting moiety comprises any one of theanti-PD-L1 antibodies obtainable from the hybridoma accessible underCNCM deposit numbers CNCM 1-4122, CNCM 1-4080 and CNCM 1-4081 asdisclosed in US 20120039906, the entire disclosures of which are herebyincorporated by reference.

In an embodiment, the targeting moiety comprises a nanobody directedagainst PD-L1 as disclosed, for example, in U.S. Pat. No. 8,907,065 andWO 2008/071447, the entire disclosures of which are hereby incorporatedby reference. In illustrative embodiments, the nanobodies against PD-L1comprise SEQ ID NOS: 394-399 of U.S. Pat. No. 8,907,065:

SEQ ID No: 394 of U.S. Pat. No. 8,907,065 (SEQ ID NO: 171):EVQLVESGGGLVQPGGSLRLSCAASGFTLDYYAIGWFRQAPGKEREWASSISSSDGSTYYADSVKGRFTISRDNAKNTVFLQMNSLKPEDTAVYSCAASQAPITIATMMKPFYDYWGQGTQVTVSS; SEQ ID No: 395 of U.S. Pat. No. 8,907,065(SEQ ID NO: 172): EVQLVESGGGLVQPGGSLRLSCAASGFTLDYYAKCWFRQAPGKEREWVSCISSSDGSTYYADSVKGRFTISRDNAKNTVYLQMNSLKPEDTAVYFCAARHGGPLTVEYFFDYWGQGTQVTVSS: SEQ ID No: 396 of U.S. Pat. No. 8,907,065 (SEQID NO: 173): EVQLVESGGGLVQPGGSLRLSCAASGFTFDYYAIGWFRQAPGKAREGVSCISGGDNSTYYADSVKGRFTISRDNAKNTVYLQMNSLKPEDTAVYYCATGGWKYCSGYDPEYIYWGQGTQVTVSS; SEQ ID No: 397 of U.S. Pat. No. 8,907,065 (SEQID NO: 174): EVQLVESGGGLVQAGGSLRLSCAASGSTFSQYDVGWYRQAPGKQRELVAFSSSGGRTIYPDSVKGRFTFSRDNTKNTVYLQMTSLKPEDTAVYYCKIDWY LNSYWGQGTQVTVSS; SEQID No: 398 of U.S. Pat. No. 8,907,065 (SEQ ID NO: 175):EVQLVESGGGLVQAGGSLRLSCAASGVDASNSAMGWYRQAPGKQREWVARITGGGLIAYTDSVKGRFTISRDNAKSTVYLQMNSLEPEDTAVYYCNTINS RDGWGQGTQVTVSS; SEQID No: 399 of U.S. Pat. No. 8,907,065 (SEQ ID NO: 176):EVQLVESGGGLVQAGGSLTISCAASGITFSDSIVSWYRRARGKQREWVAGISNGGTTKYAESVLGRFTISRDNAKNNVYLQMNGLNPEDTAVYLCKVRQY WGQGTQVTVSS.

In various embodiments, the present chimeric protein has a targetingmoiety directed against PD-L2. In some embodiments, the chimeric proteinhas a targeting moiety which selectively bind a PD-L2 polypeptide. Insome embodiments, the chimeric protein comprises one or more antibodies,antibody derivatives or formats, peptides or polypeptides, or fusionproteins that selectively bind a PD-L2 polypeptide.

In an embodiment, the targeting moiety comprises a nanobody directedagainst PD-L2 as disclosed, for example, in U.S. Pat. No. 8,907,065 andWO 2008/071447, the entire disclosures of which are hereby incorporatedby reference. In illustrative embodiments, the nanobodies against PD-1comprise SEQ ID Nos: 449-455 of U.S. Pat. No. 8,907,065:

SEQ ID No: 449 of U.S. Pat. No. 8,907,065 (SEQ ID NO: 177):EVQLVESGGGLVQAGGSLRLSCAASESTVLINAMGWYRQAPGKQRELVASISSGGSTNYADSVKGRFTISRDNAKNTVYLQMNSLKPEDTAVYYCNADVYPQDYGLGYVEGKVYYGHDYWGTGTLVTVSS; SEQ ID No: 450 of U.S. Pat. No.8,907,065 (SEQ ID NO: 178):EVQLVESGGGLVQAGGSLRLSCAASGSTFSNYVSNYAMGWGRQAPGTQRELVASISNGDTTNYADSVKGRFTISRDNAKNTVYLQMNSLKPEDTAVYYCF EHQVAGLTWGQGTQVTVSS;SEQ ID No: 451 of U.S. Pat. No. 8,907,065 (SEQ ID NO: 179):EVQLVESGGGLVQAGGSLRLSCVASGXALKIXVMGWYRQAPGKQRELVAAITSGGRTNYSDSVKGRFTISGDNAXNTVYLQMNSLKSEDTAVYYCREWNS GYPPVDYWGQGTQVTVSS;SEQ ID No: 452 of U.S. Pat. No. 8,907,065 (SEQ ID NO: 180):EVQLVESGGGLVQAGGSLRLSCAASGRTFSSGTMGWFRRAPGKEREFVASIPWSGGRTYYADSVKDRFTISRDNAQNTVFLQMNSLKPEDTAVYYCAFKE RSTGWDFASWGQGIQVTVSS;SEQ ID No: 453 of U.S. Pat. No. 8,907,065 (SEQ ID NO: 181):EVQLVESGGGLVQTGGSLRLSCAASGFTLDYYGIGWFRQAPGKEREGVSFISGSDGSTYYAESVKGRFTISRDKAKNTVYLQMNSLKPEDTAVYYCAADPWGPPSIATMTSYEYKHWGQGTQVTVSS; SEQ ID No: 454 of U.S. Pat. No. 8,907,065(SEQ ID NO: 182): EVQLVESGGGLVQPGGSLRLSCAASGFTFSTYTMIWLRRAPGKGFEWVSTIDKDGNTNYVDSVKGRFAVSRDNTKNTLYLQMNSLKPEDTAMYYCTKHGS SARGQGTRVTVSS; SEQ IDNo: 455 of U.S. Pat. No. 8,907,065 (SEQ ID NO: 183):EVQLVESGGGLVEPGGSLRLSCVASGFTFSSYDMSWVRQAPGKGLEWVSTINSGGGITYRGSVKGRFTISRDNAKNTLYLQMNSLKPEDTAVYYCENGGS SYRRGQGTQVTVSS.

In an embodiment, the targeting moiety comprises any one of theanti-PD-L2 antibodies disclosed in US2011/0271358 and WO2010/036959, theentire contents of which are hereby incorporated by reference. Inillustrative embodiments, the antibody or an antigen-binding fragmentthereof for use in the methods provided herein comprises a heavy chaincomprising an amino acid sequence selected from SEQ ID Nos: 43-47 ofUS2011/0271358:

SEQ ID No: 43 of US2011/0271358 (SEQ ID NO: 184):QVQLVQSGAELKKPGASVKMSCKASGYTFTGYTMHWVKQAPGQGLEWIGYINPRSGYTEYNQKFKDRTTLTADKSTSTAYMELSSLRSEDSAVYYCARPW FAYWGQGTLVTVSS; SEQID No: 44 of US2011/0271358 (SEQ ID NO: 185):QVQLVQSGAEVKKPGASVKMSCKASGYTFTGYTMHWVKQAPGQGLEWIGYINPRSGYTEYNQKFKDRTTLTADKSTSTAYMELSSLRSEDTAVYYCARPW FAYWGQGTLVTVSS; SEQID No: 45 of US2011/0271358 (SEQ ID NO: 186):QVQLVQSGAEVKKPGASVKMSCKASGYTFTGYTMHWVRQAPGQGLEWIGYINPRSGYTEYNQKFKDRTTLTADKSTSTAYMELSSLRSEDTAVYYCARPW FAYWGQGTLVTVSS; SEQID No: 46 of US2011/0271358 (SEQ ID NO: 187):QVQLVQSGAEVKKPGASVKVSCKASGYTFTGYTMHWVRQAPGQGLEWIGYINPRSGYTEYNQKFKDRTTLTADKSTSTAYMELSSLRSEDTAVYYCARPW FAYWGQGTLVTVSS; SEQID No: 47 of US2011/0271358 (SEQ ID NO: 188):QVQLVQSGAEVKKPGASVKVSCKASGYTFTGYTMHWVRQAPGQGLEWIGYINPRSGYTEYNQKFKDRTTITADKSTSTAYMELSSLRSEDTAVYYCARPW FAYWGQGTLVTVSS;and/or a light chain comprising an amino acid sequence selected from SEQID Nos: 48-51 of US2011/0271358:

SEQ ID No: 48 of US2011/0271358 (SEQ ID NO: 189):DIVMTQSPASLTVTPGEKVTITCKSSQSLLNSGNQKNYLTWYQQKPGQPPKLLIYWASTRESGVPDRFTGSGSGTDFTLTISSLQAEDVAVYYCQNDYSY PLTFGQGTKLEIK; SEQ IDNo: 49 of US2011/0271358 (SEQ ID NO: 190):DIVMTQSPASLSVTPGEKVTITCKSSQSLLNSGNQKNYLTWYQQKPGQPPKLLIYWASTRESGVPDRFTGSGSGTDFTLTISSLQAEDVAVYYCQNDYSY PLTFGQGTKLEIK; SEQ IDNo: 50 of US2011/0271358 (SEQ ID NO: 191):DIVMTQSPAFLSVTPGEKVTITCKSSQSLLNSGNQKNYLTWYQQKPGQPPKLLIYWASTRESGVPDRFTGSGSGTDFTLTISSLQAEDVAVYYCQNDYSY PLTFGQGTKLEIK; SEQ IDNo: 51 of US2011/0271358 (SEQ ID NO: 192):DIVMTQSPAFLSVTPGEKVTITCKSSQSLLNSGNQKNYLTWYQQKPGQPPKLLIYWASTRESGVPDRFSGSGSGTDFTLTISSLQAEDVAVYYCQNDYSY PLTFGQGTKLEIK.

In various embodiments, the targeting moieties of the invention maycomprise a sequence that targets PD-1, PD-L1, and/or PD-L2 which is atleast about 60%, at least about 61%, at least about 62%, at least about63%, at least about 64%, at least about 65%, at least about 66%, atleast about 67%, at least about 68%, at least about 69%, at least about70%, at least about 71%, at least about 72%, at least about 73%, atleast about 74%, at least about 75%, at least about 76%, at least about77%, at least about 78%, at least about 79%, at least about 80%, atleast about 81%, at least about 82%, at least about 83%, at least about84%, at least about 85%, at least about 86%, at least about 87%, atleast about 88%, at least about 89%, at least about 90%, at least about91%, at least about 92%, at least about 93%, at least about 94%, atleast about 95%, at least about 96%, at least about 97%, at least about98%, at least about 99%, or 100% identical to any of the sequencesdisclosed herein (e.g. about 60%, or about 61%, or about 62%, or about63%, or about 64%, or about 65%, or about 66%, or about 67%, or about68%, or about 69%, or about 70%, or about 71%, or about 72%, or about73%, or about 74%, or about 75%, or about 76%, or about 77%, or about78%, or about 79%, or about 80%, or about 81%, or about 82%, or about83%, or about 84%, or about 85%, or about 86%, or about 87%, or about88%, or about 89%, or about 90%, or about 91%, or about 92%, or about93%, or about 94%, or about 95%, or about 96%, or about 97%, or about98%, about 99% or about 100% sequence identity with any of the sequencesdisclosed herein).

In various embodiments, the targeting moieties of the invention maycomprise any combination of heavy chain, light chain, heavy chainvariable region, light chain variable region, complementaritydetermining region (CDR), and framework region sequences that targetPD-1, PD-L1, and/or PD-L2 as disclosed herein.

Additional antibodies, antibody derivatives or formats, peptides orpolypeptides, or fusion proteins that selectively bind or target PD-1,PD-L1 and/or PD-L2 are disclosed in WO 2011/066389, US 2008/0025980, US2013/0034559, U.S. Pat. No. 8,779,108, US 2014/0356353, U.S. Pat. No.8,609,089, US 2010/028330, US 2012/0114649, WO 2010/027827, WO2011,/066342, U.S. Pat. No. 8,907,065, WO 2016/062722, WO 2009/101611,WO2010/027827, WO 2011/066342, WO 2007/005874, WO 2001/014556,US2011/0271358, WO 2010/036959, WO 2010/077634, U.S. Pat. No. 8,217,149,US 2012/0039906, WO 2012/145493, US 2011/0318373, U.S. Pat. No.8,779,108, US 20140044738, WO 2009/089149, WO 2007/00587, WO 2016061142,WO 2016,02263, WO 2010/077634, and WO 2015/112900, the entiredisclosures of which are hereby incorporated by reference.

In various embodiments, such antigens can be found on one or more immunecells. For instance, in the treatment of various immune-modulateddiseases (including, without limitation, cancers, autoimmune diseasesand disorders and the like), targeting to immune cells will bebeneficial to the therapeutic effect. In these embodiments, the immunecells have the therapeutic receptor and the modified soluble agent caninteract with it. Illustrative immune cells include, but are not limitedto, megakaryocytes, thrombocytes, erythrocytes, mast cells, basophils,neutrophils, eosinophils, monocytes, macrophages, natural killer cells,T lymphocytes (e.g., cytotoxic T lymphocytes, T helper cells, naturalkiller T cells), B lymphocytes, plasma cells, and dendritic cells. Insome embodiments, the antigens are found on one or more hematopoieticcells in the bone marrow. In some embodiments, the antigens may also befound on hematopoietic stem cells, precursor cells, and progenitorcells.

For example, in some embodiments, the antigen recognition domainsspecifically bind to an antigen associated with B cells. Illustrative Bcell antigens of interest include, for example, CD10, CD19, CD20, CD21,CD22, CD23, CD24, CD37, CD38, CD39, CD40, CD72, CD73, CD74, CDw75,CDw76, CD77, CD78, CD79a/b, CD80, CD81, CD82, CD83, CD84, CD85, CD86,CD89, CD98, CD126, CD127, CDw130, CD138, CDw150, and B-cell maturationantigen (BCMA).

In various embodiments, the chimeric protein of the invention comprisesa targeting moiety having an antigen recognition domain thatspecifically binds to CD20. In some embodiments, the chimeric proteinfurther comprises human TNF-α as the soluble agent. In an embodiment,the human TNF-α moiety has an E146K mutation. In an embodiment, thehuman TNF-α moiety has an Y87H and an E146K mutation. In anotherembodiment, the human TNF-α moiety has an Y87H and an A145R mutation. Ina further embodiment, the human TNF-α moiety has an R32W and an S86Tmutation. In some embodiments, the antigen recognition domainsspecifically bind to an antigen associated with T cells. Illustrative Tcell antigens of interest include, for example (and inclusive of theextracellular domains, where applicable): CD8, SLAMF4, IL-2Rα,4-1BB/TNFRSF9, IL-2 R β, ALCAM, B7-1, IL-4 R, B7-H3, BLAME/SLAMFS,CEACAM1, IL-6 R, CCR3, IL-7 Rα, CCR4, CXCRI/IL-S RA, CCR5, CCR6, IL-10Rα, CCR 7, IL-I 0 R β, CCRS, IL-12 R β 1, CCR9, IL-12 R β 2, CD2, IL-13 Rα 1, IL-13, CD3, CD4, ILT2/CDS5j, ILT3/CDS5k, ILT4/CDS5d, ILT5/CDS5a,lutegrin α 4/CD49d, CDS, Integrin α E/CD103, CD6, Integrin a M/CD 11 b,CDS, Integrin α X/CD11c, Integrin β 2/CDIS, KIR/CD15S, CD27/TNFRSF7,KIR2DL1, CD2S, KIR2DL3, CD30/TNFRSFS, KIR2DL4/CD15Sd, CD31/PECAM-1,KIR2DS4, CD40 Ligand/TNFSF5, LAG-3, CD43, LAIR1, CD45, LAIR2, CDS3,Leukotriene B4-R1, CDS4/SLAMF5, NCAM-L1, CD94, NKG2A, CD97, NKG2C,CD229/SLAMF3, NKG2D, CD2F-10/SLAMF9, NT-4, CD69, NTB-A/SLAMF6, Common γChain/IL-2 R γ, Osteopontin, CRACC/SLAMF7, PD-1, CRTAM, PSGL-1, CTLA-4,RANK/TNFRSF11A, CX3CR1, CX3CL1, L-Selectin, CXCR3, SIRP β 1, CXCR4,SLAM, CXCR6, TCCR/WSX-1, DNAM-1, Thymopoietin, EMMPRIN/CD147, TIM-1,EphB6, TIM-2, Fas/TNFRSF6, TIM-3, Fas Ligand/TNFSF6, TIM-4, FcγRIII/CD16, TIM-6, TNFR1/TNFRSF1A, Granulysin, TNF RIII/TNFRSF1B, TRAILRI/TNFRSFIOA, ICAM-1/CD54, TRAIL R2/TNFRSF10B, ICAM-2/CD102,TRAILR3/TNFRSF10C, IFN-γR1, TRAILR4TTNFRSF10D, IFN-γ R2, TSLP, IL-1 R1and TSLP R.

In some embodiments, the present chimeric protein comprises a targetingmoiety directed against CD3 expressed on T cells. In some embodiments,the chimeric protein comprises a targeting moiety which selectively binda CD3 polypeptide. In some embodiments, the chimeric protein comprisesone or more antibodies, antibody derivatives or formats, peptides orpolypeptides, or fusion proteins that selectively bind a CD3polypeptide.

In an embodiment, the targeting moiety comprises the anti-CD3 antibodymuromonab-CD3 (aka Orthoclone OKT3), or fragments thereof. Muromonab-CD3is disclosed in U.S. Pat. No. 4,361,549 and Wilde et al. (1996)51:865-894, the entire disclosures of which are hereby incorporated byreference. In illustrative embodiments, muromonab-CD3 or anantigen-binding fragment thereof for use in the methods provided hereincomprises a heavy chain comprising the amino acid sequence of:

SEQ ID NO: 193 QVQLQQSGAELARPGASVKMSCKASGYTFTRYTMHWVKQRPGQGLEWIGYINPSRGYTNYNQKFKDKATLTTDKSSSTAYMQLSSLTSEDSAVYYCARYYDDHYCLDYWGQGTTLTVSSAKTTAPSVYPLAPVCGGTTGSSVTLGCLVKGYFPEPVTLTWNSGSLSSGVHTFPAVLQSDLYTLSSSVTVTSSTWPSQSITCNVAHPASSTKVDKKIEPRPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCWVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK;and/or a light chain comprising the amino acid sequence of:

SEQ ID NO: 194 QIVLTQSPAIMSASPGEKVTMTCSASSSVSYMNWYQQKSGTSPKRWIYDTSKLASGVPAHFRGSGSGTSYSLTISGMEAEDAATYYCQQWSSNPFTFGSGTKLEINRADTAPTVSIFPPSSEQLTSGGASVVCFLNNFYPKDINVKWKIDGSERQNGVLNSWTDQDSKDSTYSMSSTLTLTKDEYERHNSYTCEATHKTS TSPIVKSFNRNEC.

In an embodiment, the targeting moiety comprises the anti-CD3 antibodyotelixizumab, or fragments thereof. Otelixizumab is disclosed in U.S.Patent Publication No. 20160000916 and Chatenoud et al. (2012)9:372-381, the entire disclosures of which are hereby incorporated byreference. In illustrative embodiments, otelixizumab or anantigen-binding fragment thereof for use in the methods provided hereincomprises a heavy chain comprising the amino acid sequence of:

SEQ ID NO: 195 EVQLLESGGGLVQPGGSLRLSCAASGFTFSSFPMAWVRQAPGKGLEWVSTISTSGGRTYYRDSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCAKFRQYSGGFDYWGQGTLVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYASTYRWSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK;and/or a light chain comprising the amino acid sequence of:

SEQ ID NO: 196 DIQLTQPNSVSTSLGSTVKLSCTLSSGNIENNYVHWYQLYEGRSPTTMIYDDDKRPDGVPDRFSGSIDRSSNSAFLTIHNVAIEDEAIYFCHSYVSSFNVFGGGTKLTVLRQPKAAPSVTLFPPSSEELQANKATLVCLISDFYPGAVTVAWKADSSPVKAGVETTTPSKQSNNKYAASSYLSLTPEQWKSHRSYSCQVT HEGSTVEKTVAPTECS

In an embodiment, the targeting moiety comprises the anti-CD3 antibodyteplizumab (AKA MGA031 and hOKT3γ1(Ala-Ala)), or fragments thereof.Teplizumab is disclosed in Chatenoud et al. (2012) 9:372-381, the entiredisclosures of which are hereby incorporated by reference. Inillustrative embodiments, teplizumab or an antigen-binding fragmentthereof for use in the methods provided herein comprises a heavy chaincomprising the amino acid sequence of:

SEQ ID NO: 197 QVQLVQSGGGVVQPGRSLRLSCKASGYTFTRYTMHWVRQAPGKGLEWIGYINPSRGYTNYNQKVKDRFTISRDNSKNTAFLQMDSLRPEDTGVYFCARYYDDHYCLDYWGQGTPVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPEAAGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRWSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK;and/or a light chain comprising the amino acid sequence of:

SEQ ID NO: 198 DIQMTQSPSSLSASVGDRVTITCSASSSVSYMNWYQQTPGKAPKRWIYDTSKLASGVPSRFSGSGSGTDYTFTISSLQPEDIATYYCQQWSSNPFTFGQGTKLQITRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQG LSSPVTKSFNRGEC.

In an embodiment, the targeting moiety comprises the anti-CD3 antibodyvisilizumab (AKA Nuvion®; HuM291), or fragments thereof. Visilizumab isdisclosed in U.S. Pat. No. 5,834,597 and WO2004052397, and Cole et al.,Transplantation (1999) 68:563-571, the entire disclosures of which arehereby incorporated by reference. In illustrative embodiments,visilizumab or an antigen-binding fragment thereof for use in themethods provided herein comprises a heavy chain variable regioncomprising the amino acid sequence of:

SEQ ID NO: 199 QVQLVQSGAEVKKPGASVKVSCKASGYTFISYTMHWVRQAPGQGLEWMGYINPRSGYTHYNQKLKDKATLTADKSASTAYMELSSLRSEDTAVYYCARSAYYDYDGFAYWGQGTLVTVSS;and/or a light chain variable region comprising the amino acid sequenceof:

SEQ ID NO: 200 DIQMTQSPSSLSASVGDRVTITCSASSSVSYMNWYQQKPGKAPKRLIYDTSKLASGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQWSSNPPTFG GGTKVEIK.

In an embodiment, the targeting moiety comprises the anti-CD3 antibodyforalumab (aka NI-0401), or fragments thereof. In various embodiments,the targeting moiety comprises any one of the anti-CD3 antibodiesdisclosed in US20140193399, U.S. Pat. No. 7,728,114, US20100183554, andU.S. Pat. No. 8,551,478, the entire disclosures of which are herebyincorporated by reference.

In illustrative embodiments, the anti-CD3 antibody or an antigen-bindingfragment thereof for use in the methods provided herein comprises aheavy chain variable region comprising the amino acid sequence of SEQ IDNos: 2 and 6 of U.S. Pat. No. 7,728,114:

SEQ ID No: 2 of U.S. Pat. No. 7,728,114 (SEQ ID NO: 201):QVQLVESGGGVVQPGRSLRLSCAASGFKFSGYGMHWVRQAPGKGLEWVAVIWYDGSKKYYVDSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCAR QMGYWHFDLWGRGTLVTVSS;SEQ ID No: 6 of U.S. Pat. No. 7,728,114 (SEQ ID NO: 202):QVQLVQSGGGVVQSGRSLRLSCAASGFKFSGYGMHWVRQAPGKGLEWVAVIWYDGSKKYYVDSVKGRFTISRDNSKNTLYLQMNSLRGEDTAVYYCAR QMGYWHFDLWGRGTLVTVSS;and/or a light chain variable region comprising the amino acid sequenceof SEQ ID NOs 4 and 8 of U.S. Pat. No. 7,728,114:

SEQ ID No: 4 of U.S. Pat. No. 7,728,114 (SEQ ID NO: 203):EIVLTQSPATLSLSPGERATLSCRASQSVSSYLAWYQQKPGQAPRLLIYDASNRATGIPARFSGSGSGTDFTLTISSLEPEDFAVYYCQQRSNWPPLT FGGGTKVEIK; SEQ ID No:8 of U.S. Pat. No. 7,728,114 (SEQ ID NO: 204):EIVLTQSPATLSLSPGERATLSCRASQSVSSYLAWYQQKPGQAPRLLIYDASNRATGIPARFSGSGSGTDFTLTISSLEPEDFAVYYCQQRSNWPPLT FGGGTKVEIK;

In an embodiment, the targeting moiety comprises a heavy chain variableregion comprising the amino acid sequence of SEQ ID NO:2 of U.S. Pat.No. 7,728,114 and a light chain variable region comprising the aminoacid sequence of SEQ ID NO:4 of U.S. Pat. No. 7,728,114. In anembodiment, the targeting moiety comprises any one of the anti-CD3antibodies disclosed in US2016/0168247, the entire contents of which arehereby incorporated by reference. In illustrative embodiments, theantibody or an antigen-binding fragment thereof for use in the methodsprovided herein comprises a heavy chain comprising an amino acidsequence selected from SEQ ID Nos: 6-9 of US2016/0168247:

SEQ ID No: 6 of US2016/0168247 (SEQ ID NO: 205):EVKLVESGGGLVQPGGSLRLSCAASGFTFNTYAMNWVRQAPGKGLEWVARIRSKYNNYATYYADSVKDRFTISRDDSKSSLYLQMNNLKTEDTAMYYCVRHGNFGNSYVSWFAYWGQGTLVTVSS; SEQ ID No: 7 of US2016/0168247 (SEQ ID NO:206): EVKLVESGGGLVKPGRSLRLSCAASGFTFNTYAMNWVRQAPGKGLEWVARIRSKYNNYATYYADSVKDRFTISRDDSKSILYLQMNNLKTEDTAMYYCVRHGNFGNSYVSWFAYWGQGTLVTVSS; SEQ ID No: 8 of US2016/0168247 (SEQ ID NO:207): EVKLVESGGGLVKPGRSLRLSCAASGFTFNTYAMNWVRQAPGKGLEWVARIRSKYNNYATYYADSVKDRFTISRDDSKSILYLQMNSLKTEDTAMYYCVRHGNFGNSYVSWFAYWGQGTLVTVSS; SEQ ID No: 9 of US2016/0168247 (SEQ ID NO:208): EVKLVESGGGLVKPGRSLRLSCAASGFTFNTYAMNWVRQAPGKGLEWVARIRSKYNNYATYYADSVKDRFTISRDDSKSILYLQMNSLKTEDTAMYYCVRHGNFGNSYVSWFAYWGQGTMVTVSS;and/or a light chain comprising an amino acid sequence selected from SEQID Nos: 10-12 of US2016/0168247:

SEQ ID No: 10 of US2016/0168247 (SEQ ID NO: 209):QAVVTQEPSFSVSPGGTVTLTCRSSTGAVTTSNYANWVQQTPGQAFRGLIGGTNKRAPGVPARFSGSLIGDKAALTITGAQADDESIYFCALWYSNLWVF GGGTKLTVL; SEQ ID No:11 of US2016/0168247 (SEQ ID NO: 210):QAVVTQEPSFSVSPGGTVTLTCRSSTGAVTTSNYANWVQQTPGQAFRGLIGGTNKRAPGVPARFSGSILGNKAALTITGAQADDESIYFCALWYSNLWVF GGGTKLTVL; SEQ ID No:12 of US2016/0168247 (SEQ ID NO: 211):QAVVTQEPSFSVSPGGTVTLTCRSSTGAVTTSNYANWVQQTPGQAFRGLIGGTNKRAPGVPARFSGSILGNKAALTITGAQADDESDYYCALWYSNLWVF GGGTKLTVL.

In an embodiment, the targeting moiety comprises any one of the anti-CD3antibodies disclosed in US2015/0175699, the entire contents of which arehereby incorporated by reference. In illustrative embodiments, theantibody or an antigen-binding fragment thereof for use in the methodsprovided herein comprises a heavy chain comprising an amino acidsequence selected from SEQ ID No: 9 of US2015/0175699:

SEQ ID No: 9 of US2015/0175699 (SEQ ID NO: 212):QVQLVQSGSELKKPGASVKMSCKASGYTFTRYTMHWVRQAPGKGLEWIGYINPSRGYTNYNQKFKDRATLTTDKSTSTAYMQLSSLRSEDTAVYYCAR YYDDHYSLDYWGQGTLVTVSS;and/or a light chain comprising an amino acid sequence selected from SEQID No: 10 of US2015/0175699:

SEQ ID No: 10 of US2015/0175699 (SEQ ID NO: 213):QIVLTQSPATLSLSPGERATMSCSASSSVSYMNWYQQKPGKAPKRWIYDTSKLASGVPSRFRGSGSGTDYTLTISSLQPEDFATYYCQQWSSNPFTFGGG TKVEIK.

In an embodiment, the targeting moiety comprises any one of the anti-CD3antibodies disclosed in U.S. Pat. No. 8,784,821, the entire contents ofwhich are hereby incorporated by reference. In illustrative embodiments,the antibody or an antigen-binding fragment thereof for use in themethods provided herein comprises a heavy chain comprising an amino acidsequence selected from SEQ ID Nos: 2, 18, 34, 50, 66, 82, 98 and 114 ofU.S. Pat. No. 8,784,821:

SEQ ID No: 2 of U.S. Pat. No. 8,784,821 (SEQ ID NO: 214):ELQLVESGGGVVQPGRSLRLSCAASGFTFSSYGMHWVRQAPGKGLEWVAVISYDGSNKYYADSVKGRFTISRDNSKNTLYLQMNSLRSEDTAVYYCARLSPYCTNGVCWDAFDIWGQGTMVTVSS; SEQ ID No: 18 of U.S. Pat. No. 8,784,821(SEQ ID NO: 215): ELQLVESGGGLVKPGRSLRLSCTASGFTFGDYAMSWFRQAPGKGLEWVGFIRSKAYGGTTEYAASVKGRFTISRDDSKSIAYLQMNSLKTEDTAVYYCTPQLWLLQDAFDIWGQGTMVTVSS; SEQ ID No: 34 of U.S. Pat. No. 8,784,821 (SEQID NO: 216): ELQLVESGPGLVKPSGTLSLTCAVSGGSISSRNWWSWVRQPPGKGLEWIGDIYHSGSTNYNPSLKSRVTISVDKSKNQFSLKLSSVTAADTAVYYCASGYTSCRDAFDIWGQGTMVTVSS; SEQ ID No: 50 of U.S. Pat. No. 8,784,821 (SEQ IDNO: 217): ELQLVEWGAGLLKPSETLSLTCAVYGGSFSGYYWSWIRQPPGKGLEWIGEINHSGSTNYNPSLKSRVTISVDTSKNQFSLKLSSVTAADTAVYYCARGRGRFLGWLLGGSNWFDPWGQGTLVTVSS; SEQ ID No: 66 of U.S. Pat. No. 8,784,821(SEQ ID NO: 218): ELQLVEWGAGLLKPSETLSLTCAVYGGSFSGYYWSWIRQPPGKGLEWIGEINHSGSTNYNPSLKSRVTISVDTSKNQFSLKLSSVTAADTAVYYCARGP DRMGHGFDIWGQGTMVTVSS;SEQ ID No: 82 of U.S. Pat. No. 8,784,821 (SEQ ID NO: 219):ELQLVESGPGLVKPSQTLSLTCAISGDSVSSNSAAWNWIRQSPSRGLEWLGRTYYRSKWYNDYAVSVKSRITINPDTSKNQFSLQLNSVTPEDTAVYYCARDRRRIAARQYYGMDVWGQGTTVTVSS; SEQ ID No: 98 of U.S. Pat. No. 8,784,821(SEQ ID NO: 220): ELQLVESGGGLVQPGGSLRLSCAASGFTFSSYAMGWVRQAPGKGLEWVSAVSGSGGSTYYADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCAKAKFLGHYYGMDVWGQGTTVTVSS; SEQ ID No: 114 of U.S. Pat. No. 8,784,821 (SEQID NO: 221): ELQLVESGPVLVKPTDTLTLTCTVSGFSLNNPRMGVSWIRQPPGKTLEWLAHIFPSDAKAHSASLKSRLTISKDTSKSQVVPTMTNMDPVDTATYYCARILGEYYPPAWFDPWGQGTLVTVSS;and/or a light chain comprising an amino acid sequence selected from SEQID Nos: 10, 26, 42, 58, 74, 90, 106 and 122 of U.S. Pat. No. 8,784,821:

SEQ ID No: 10 of U.S. Pat. No. 8,784,821 (SEQ ID NO: 222):ELQMTQSPSSLSASVGDRVSITCRASQTISNYLNWYQLKPGKAPKLLIYAASTLQSEVPTRFSGSGSGTDFTLTISGLHPEDFATYYCQQFNSYPRTF GQGTKVEIK; SEQ ID No:26 of U.S. Pat. No. 8,784,821 (SEQ ID NO: 223):ELQMTQSPSSLSASVGDRVTITCRASQGISNYLAWYQQKPGKVPKLLIYAASTLQSGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQSYSTPPTF GQGTKLEIK; SEQ ID No:42 of U.S. Pat. No. 8,784,821 (SEQ ID NO: 224):ELVMTQSPSSLSASVGDRVTITCRASQGIGNYLAWYQQKPGQPPKMLIYWASIRESGVPDRFSGSGSGTDFTLTISSLQAEDVAVYYCQQYYSNPQ TFGQGTKVEIK; SEQ ID No:58 of U.S. Pat. No. 8,784,821 (SEQ ID NO: 225):ELVMTQSPSSLSASVGDRVTITCRASQGISNYLNWYQQKPGKAPKLLIYDASNLETGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQSYSTPYTF GQGTKVDIK; SEQ ID No:74 of U.S. Pat. No. 8,784,821 (SEQ ID NO: 226):ELQMTQSPSSLSASVGDRVTITCRASQSISSYLNWYQQKSGKAPKLLIYAASSLQSGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQSYSSPWTF GQGTKVEIK; SEQ ID No:90 of U.S. Pat. No. 8,784,821 (SEQ ID NO: 227):ELVLTQSPGTLSLSPGERATLSCRASQSVSSNYLAWYQQKPGQAPRLLIYGASSRATGIPDRFSGSGSGTDFTLTISSLQPEDVATYYCQKYNSAPLT FGGGTKVEIK; SEQ ID No:106 of U.S. Pat. No. 8,784,821 (SEQ ID NO: 228):ELQMTQSPSSLSASVGDRVTITCRASQSISSYLNWYQQKPGKAPKLLIYAASSLQSGVPSRFSGSGSGTEFTLTISSLQPEDFATYYCLQHNAYPYTF GQGTKVEIK; SEQ ID No:122 of U.S. Pat. No. 8,784,821 (SEQ ID NO: 229):ELVMTQSPDSLAVSLGERATINCKSSQSVLYSSNNKNYLAWYQQKPGQPPKLLIYWASTRESGVPDRFSGSGSGTDFTLTISSLQAEDVAVYYCQQYL KIPYTFGQGTKVEIK.

In an embodiment, the targeting moiety comprises any one of the anti-CD3binding constructs disclosed in US20150118252, the entire contents ofwhich are hereby incorporated by reference. In illustrative embodiments,the antibody or an antigen-binding fragment thereof for use in themethods provided herein comprises a heavy chain comprising an amino acidsequence selected from SEQ ID Nos: 6 and 86 of US20150118252:

SEQ ID No: 6 of US20150118252 (SEQ ID NO: 230):QVQLVQSGAEVKKPGASVKVSCKASGYTFTRYTMHWVRQAPGQGLEWMGYINPSRGYTNYNQKFKDRVTMTTDTSISTAYMELSRLRSDDTAVYYCARYYDDHYCLDYWGQGTLVTVSS; SEQ ID No: 86 of US20150118252 (SEQ ID NO: 231):QVQLVQSGAEVKKPGASVKVSCKASGYTFTRYTMHWVRQAPGQGLEWMGYINPSRGYTNYNQKFKDRVTMTTDTSISTAYMELSRLRSDDTAVYYCARYYDDHYSLDYWGQGTLVTVSS;and/or a light chain comprising an amino acid sequence selected from SEQID No: 3 of US2015/0175699:

SEQ ID No: 3 of US20150118252 (SEQ ID NO: 232):EIVLTQSPATLSLSPGERATLSCSASSSVSYMNWYQQKPGQAPRLLIYDTSKLASGVPAHFRGSGSGTDYTLTISSLEPEDFAVYYCQQWSSNPFTFG QGTKVEIK.

In an embodiment, the targeting moiety comprises any one of the anti-CD3binding proteins disclosed in US2016/0039934, the entire contents ofwhich are hereby incorporated by reference. In illustrative embodiments,the antibody or an antigen-binding fragment thereof for use in themethods provided herein comprises a heavy chain comprising an amino acidsequence selected from SEQ ID Nos: 6-9 of US2016/0039934:

SEQ ID No: 6 of US2016/0039934 (SEQ ID NO: 233):EVQLVESGGGLVQPGGSLRLSCAASGFTFSTYAMNWVRQAPGKGLEWVGRIRSKYNNYATYYADSVKDRFTISRDDSKNSLYLQMNSLKTEDTAVYYCARHGNFGNSYVSWFAYWGQGTLVTVSS; SEQ ID No: 7 of US2016/0039934 (SEQ ID NO:234): EVQLVESGGGLVQPGGSLRLSCAASGFTFSTYAMNWVRQAPGKGLEWVGRIRSKYNNYATYYADSVKDRFTISRDDSKNSLYLQMNSLKTEDTAVYYCARHGNFGNSYVSWFAYWGQGTLVTVSS; SEQ ID No: 8 of US2016/0039934 (SEQ ID NO:235): EVQLVESGGGLVQPGGSLRLSCAASGFTFSTYAMNWVRQAPGKGLEWVGRIRSKYNNYATYYADSVKDRFTISRDDSKNSLYLQMNSLKTEDTAVYYCARHGNFGNSYVSYFAYWGQGTLVTVSS; SEQ ID No: 9 of US2016/0039934 (SEQ ID NO:236): EVQLVESGGGLVQPGGSLRLSCAASGFTFSTYAMNWVRQAPGKGLEWVGRIRSKYNNYATYYADSVKDRFTISRDDSKNSLYLQMNSLKTEDTAVYYCARHGNFGNSYVSHFAYWGQGTLVTVSS;and/or a light chain comprising an amino acid sequence selected from SEQID Nos: 1-4 of US2016/0039934:

SEQ ID No: 1 of US2016/0039934 (SEQ ID NO: 237):DIQMTQSPSSLSASVGDRVTITCRSSTGAVTTSNYANWVQQKPGKAPKGLIGGTNKRAPGVPSRFSGSLIGDKATLTISSLQPEDFATYYCALWYSNL WVFGQGTKVEIK; SEQ IDNo: 2 of US2016/0039934 (SEQ ID NO: 238):DIQMTQSPSSLSASVGDRVTITCRSSTGAVTTSNYANWVQQKPGKAPKGLIGGTNKRAPGVPARFSGSGSGTDFTLTISSLQPEDFATYYCALWYSNL WVFGQGTKVEIK; SEQ IDNo: 3 of US2016/0039934 (SEQ ID NO: 239):DIQMTQSPSSLSASVGDRVTITCRSSTGAVTTSNYANWVQQKPGKAPKALIGGTNKRAPGVPSRFSGSLIGDKATLTISSLQPEDFATYYCALWYSNL WVFGQGTKVEIK; SEQ IDNo: 4 of US2016/0039934 (SEQ ID NO: 240):DIQMTQSPSSLSASVGDRVTITCRSSTGAVTTSNYANWVQQKPGKAPKGLIGGTNKRAPGVPSRFSGSLIGDKATLTISSLQPEDFATYYCALWYSNL WVFGQGTKVEIK;

In various embodiments, the targeting moieties of the invention maycomprise a sequence that targets CD3 which is at least about 60%, atleast about 61%, at least about 62%, at least about 63%, at least about64%, at least about 65%, at least about 66%, at least about 67%, atleast about 68%, at least about 69%, at least about 70%, at least about71%, at least about 72%, at least about 73%, at least about 74%, atleast about 75%, at least about 76%, at least about 77%, at least about78%, at least about 79%, at least about 80%, at least about 81%, atleast about 82%, at least about 83%, at least about 84%, at least about85%, at least about 86%, at least about 87%, at least about 88%, atleast about 89%, at least about 90%, at least about 91%, at least about92%, at least about 93%, at least about 94%, at least about 95%, atleast about 96%, at least about 97%, at least about 98%, at least about99%, or 100% identical to any of the sequences disclosed herein (e.g.about 60%, or about 61%, or about 62%, or about 63%, or about 64%, orabout 65%, or about 66%, or about 67%, or about 68%, or about 69%, orabout 70%, or about 71%, or about 72%, or about 73%, or about 74%, orabout 75%, or about 76%, or about 77%, or about 78%, or about 79%, orabout 80%, or about 81%, or about 82%, or about 83%, or about 84%, orabout 85%, or about 86%, or about 87%, or about 88%, or about 89%, orabout 90%, or about 91%, or about 92%, or about 93%, or about 94%, orabout 95%, or about 96%, or about 97%, or about 98%, about 99% or about100% sequence identity with any of the sequences disclosed herein).

In various embodiments, the targeting moieties of the invention maycomprise any combination of heavy chain, light chain, heavy chainvariable region, light chain variable region, complementaritydetermining region (CDR), and framework region sequences that target CD3as disclosed herein. In various embodiments, the targeting moieties ofthe invention may comprise any heavy chain, light chain, heavy chainvariable region, light chain variable region, complementaritydetermining region (CDR), and framework region sequences of theCD3-specific antibodies including, but not limited to, X35-3, VIT3,BMA030 (BW264/56), CLB-T3/3, CRIS7, YTH12.5, FI 11-409, CLB-T3.4.2,TR-66, WT32, SPv-T3b, 11D8, XIII-141, XIII-46, XIII-87, 12F6,T3/RW2-808, T3/RW2-4B6, OKT3D, M-T301, SMC2, WT31 and F101.01. TheseCD3-specific antibodies are well known in the art and, inter alia,described in Tunnacliffe (1989), Int. Immunol. 1, 546-550, the entiredisclosures of which are hereby incorporated by reference.

Additional antibodies, antibody derivatives or formats, peptides orpolypeptides, or fusion proteins that selectively bind or target CD3 aredisclosed in US Patent Publication No. 2016/0000916, U.S. Pat. Nos.4,361,549, 5,834,597, 6,491,916, 6,406,696, 6,143,297, 6,750,325 andInternational Publication No. WO 2004/052397, the entire disclosures ofwhich are hereby incorporated by reference.

By way of further example, in some embodiments, the antigen recognitiondomains specifically bind to an antigen associated with Natural Killercells. Illustrative Natural Killer cell antigens of interest include,for example 2B4/SLAMF4, KIR2DS4, CD155/PVR, KIR3DL1, CD94, LMIR1/CD300A,CD69, LMIR2/CD300c, CRACC/SLAMF7, LMIR3/CD300LF, DNAM-1, LMIR5/CD300LB,Fc-epsilon RII, LMIR6/CD300LE, Fc-γ RI/CD64, MICA, Fc-γ RIIB/CD32b,MICB, Fc-γ RIIC/CD32c, MULT-1, Fc-γ RIIA/CD32a, Nectin-2/CD112, Fc-γRIII/CD16, NKG2A, FcRH1/IRTA5, NKG2C, FcRH2/IRTA4, NKG2D, FcRH4/IRTA1,NKp30, FcRH5/IRTA2, NKp44, Fc-Receptor-like 3/CD16-2, NKp46/NCR1,NKp80/KLRF1, NTB-A/SLAMF6, Rae-1, Rae-1 α, Rae-1 β, Rae-1 delta, H60,Rae-1 epsilon, ILT2/CD85j, Rae-1 γ, ILT3/CD85k, TREM-1, ILT4/CD85d,TREM-2, ILT5/CD85a, TREM-3, KIR/CD158, TREML1/TLT-1, KIR2DL1, ULBP-1,KIR2DL3, ULBP-2, KIR2DL4/CD158d and ULBP-3.

Also, in some embodiments, the antigen recognition domains specificallybind to an antigen associated with macrophages/monocytes. Illustrativemacrophages/monocyte antigens of interest include, for exampleB7-1/CD80, ILT4/CD85d, B7-H1, ILT5/CD85a, Common β Chain, Integrin a4/CD49d, BLAME/SLAMF8, Integrin α X/CDIIc, CCL6/C10, Integrin β 2/CD18,CD155/PVR, Integrin β 3/CD61, CD31/PECAM-1, Latexin, CD36/SR-B3,Leukotriene B4 R1, CD40/TNFRSF5, LIMPIIISR-B2, CD43, LMIR1/CD300A, CD45,LMIR2/CD300c, CD68, LMIR3/CD300LF, CD84/SLAMF5, LMIR5/CD300LB, CD97,LMIR6/CD300LE, CD163, LRP-1, CD2F-10/SLAMF9, MARCO, CRACC/SLAMF7, MD-1,ECF-L, MD-2, EMMPRIN/CD147, MGL2, Endoglin/CD105, Osteoactivin/GPNMB,Fc-γ RI/CD64, Osteopontin, Fc-γ RIIB/CD32b, PD-L2, Fc-γ RIIC/CD32c,Siglec-3/CD33, Fc-γ RIIA/CD32a, SIGNR1/CD209, Fc-γ RIII/CD16, SLAM,GM-CSF R α, TCCR/WSX-1, ICAM-2/CD102, TLR3, IFN-γ RI, TLR4, IFN-gammaR2, TREM-I, IL-I RII, TREM-2, ILT2/CD85j, TREM-3, ILT3/CD85k,TREM1/TLT-1, 2B4/SLAMF 4, IL-10 R α, ALCAM, IL-10 R β,AminopeptidaseN/ANPEP, ILT2/CD85j, Common β Chain, ILT3/CD85k, ClqR1/CD93, ILT4/CD85d, CCR1, ILT5/CD85a, CCR2, Integrin a 4/CD49d, CCR5,Integrin α M/CDII b, CCR8, Integrin a X/CDIIc, CD155/PVR, Integrin β2/CD18, CD14, Integrin β 3/CD61, CD36/SR-B3, LAIR1, CD43, LAIR2, CD45,Leukotriene B4-R1, CD68, LIMPIIISR-B2, CD84/SLAMF5, LMIR1/CD300A, CD97,LMIR2/CD300c, CD163, LMIR3/CD300LF, Coagulation Factor III/TissueFactor, LMIR5/CD300LB, CX3CR1, CX3CL1, LMIR6/CD300LE, CXCR4, LRP-1,CXCR6, M-CSF R, DEP-1/CD148, MD-1, DNAM-1, MD-2, EMMPRIN/CD147, MMR,Endoglin/CD105, NCAM-L1, Fc-γ RI/CD64, PSGL-1, Fc-γ RIIIICD16, RP105,G-CSF R, L-Selectin, GM-CSF R α, Siglec-3/CD33, HVEM/TNFRSF14, SLAM,ICAM-1/CD54, TCCR/WSX-1, ICAM-2/CD102, TREM-I, IL-6 R, TREM-2,CXCRI/IL-8 RA, TREM-3 and TREMLI/TLT-1.

Also, in some embodiments, the antigen recognition domains specificallybind to an antigen associated with dendritic cells. Illustrativedendritic cell antigens of interest include, for example CLEC9A,CD36/SRB3, LOX-1/SR-E1, CD68, MARCO, CD163, SR-A1/MSR, CD5L, SREC-1,CL-PI/COLEC12, SREC-II, LIMPIIISRB2, RP105, TLR4, TLR1, TLR5, TLR2,TLR6, TLR3, TLR9, 4-IBB Ligand/TNFSF9, IL-12/IL-23 p40,4-Amino-1,8-naphthalimide, ILT2/CD85j, CCL21/6Ckine, ILT3/CD85k,8-oxo-dG, ILT4/CD85d, 8D6A, ILT5/CD85a, A2B5, lutegrin α 4/CD49d, Aag,Integrin β 2/CD18, AMICA, Langerin, B7-2/CD86, Leukotriene B4 RI, B7-H3,LMIR1/CD300A, BLAME/SLAMF8, LMIR2/CD300c, Clq R1/CD93, LMIR3/CD300LF,CCR6, LMIR5/CD300LB CCR7, LMIR6/CD300LE, CD40/TNFRSF5, MAG/Siglec-4-a,CD43, MCAM, CD45, MD-1, CD68, MD-2, CD83, MDL-1/CLEC5A, CD84/SLAMF5,MMR, CD97, NCAMLI, CD2F-10/SLAMF9, Osteoactivin GPNMB, Chern 23, PD-L2,CLEC-1, RP105, CLEC-2, CLEC-8, Siglec-2/CD22, CRACC/SLAMF7,Siglec-3/CD33, DC-SIGN, Siglec-5, DC-SIGNR/CD299, Siglec-6, DCAR,Siglec-7, DCIR/CLEC4A, Siglec-9, DEC-205, Siglec-10, Dectin-1/CLEC7A,Siglec-F, Dectin-2/CLEC6A, SIGNR1/CD209, DEP-1/CD148, SIGNR4, DLEC,SLAM, EMMPRIN/CD147, TCCR/WSX-1, Fc-γ R1/CD64, TLR3, Fc-γ RIIB/CD32b,TREM-1, Fc-γ RIIC/CD32c, TREM-2, Fc-γ RIIA/CD32a, TREM-3, Fc-γRIII/CD16, TREML1/TLT-1, ICAM-2/CD102 and Vanilloid R1.

In some embodiments, the antigen recognition domains specifically bindto an antigen associated with megakaryocytes and/or thrombocytes.Illustrative megakaryocyte and/or thrombocyte antigens of interestinclude, for example, GP IIb/IIIa, GPIb, vWF, PF4, and TSP.

In some embodiments, the antigen recognition domains specifically bindto an antigen associated with erythrocytes. In an embodiment, theantigen recognition domains specifically bind to an antigen associatedwith erythroid progenitor and/or precursor cells. Illustrative erythroidprogenitor and/or precursor cells include, for example, the earliesterythroid progenitor, the BFU-E (burst forming unit-erythroid) and thelater-stage erythroid progenitor cell, CFU-E (colony-formingunits-erythroid). Illustrative antigens associated with erythroidprogenitor and/or precursor cells include, but are not limited to, CD34,CD36, CD38, CD41a (platelet glycoprotein IIb/IIIa), CD41b (GPIIb), CD71(transferrin receptor), CD105, glycophorin A, glycophorin C, c-kit,HLA-DR, H2 (MHC-II), and Rhesus antigens.

In some embodiments, the antigen recognition domains specifically bindto an antigen associated with mast cells. Illustrative mast cellsantigens of interest include, for example, SCFR/CD117, Fc_(ε)RI, CD2,CD25, CD35, CD88, CD203c, C5R1, CMAI, FCERIA, FCER2, TPSABI.

In some embodiments, the antigen recognition domains specifically bindto an antigen associated with basophils. Illustrative basophils antigensof interest include, for example, Fc_(ε)RI, CD203c, CD123, CD13, CD107a,CD107b, and CD164.

In some embodiments, the antigen recognition domains specifically bindto an antigen associated with neutrophils. Illustrative neutrophilsantigens of interest include, for example, 7D5, CD10/CALLA, CD13, CD16(FcRIII), CD18 proteins (LFA-1, CR3, and p150,95), CD45, CD67, andCD177.

In some embodiments, the antigen recognition domains specifically bindto an antigen associated with eosinophils. Illustrative eosinophilsantigens of interest include, for example, CD35, CD44 and CD69.

In various embodiments, the antigen recognition domain may bind to anyappropriate antigen or cell surface markers known by the skilledartisan. In some embodiments, the antigen or cell surface marker is atissue-specific marker. Illustrative tissue-specific markers include,but are not limited to, endothelial cell surface markers such as ACE,CD14, CD34, CDH5, ENG, ICAM2, MCAM, NOS3, PECAMI, PROCR, SELF, SELP,TEK, THBD, VCAMI, VWF; smooth muscle cell surface markers such as ACTA2,MYHIO, MYHI 1, MYH9, MYOCD; fibroblast (stromal) cell surface markerssuch as ALCAM, CD34, COLIAI, COL1A2, COL3A1, PH-4; epithelial cellsurface markers such as CDID, K6IRS2, KRTIO, KRT13, KRT17, KRT18, KRT19,KRT4, KRT5, KRT8, MUCI, TACSTDI; and adipocyte surface markers such asADIPOQ, FABP4, and RETN.

In various embodiments, the antigen recognition domains specificallybind to a target (e.g. antigen, receptor) which is part of anon-cellular structure. In some embodiments, the antigen or receptor isnot an integral component of an intact cell or cellular structure. Insome embodiments, the antigen or receptor is an extracellular antigen orreceptor. In some embodiments, the target is a non-proteinaceous,non-cellular marker, including, without limitation, nucleic acids,inclusive of DNA or RNA, such as, for example, DNA released fromnecrotic tumor cells or extracellular deposits such as cholesterol.

In some embodiments, the target (e.g. antigen, receptor) of interest ispart of the non-cellular component of the stroma or the extracellularmatrix (ECM) or the markers associated therewith. As used herein, stromarefers to the connective and supportive framework of a tissue or organ.Stroma may include a compilation of cells such asfibroblasts/myofibroblasts, glial, epithelia, fat, immune, vascular,smooth muscle, and immune cells along with the extracellular matrix(ECM) and extracellular molecules. In various embodiments, the target(e.g. antigen, receptor) of interest is part of the non-cellularcomponent of the stroma such as the extracellular matrix andextracellular molecules. As used herein, the ECM refers to thenon-cellular components present within all tissues and organs. The ECMis composed of a large collection of biochemically distinct componentsincluding, without limitation, proteins, glycoproteins, proteoglycans,and polysaccharides. These components of the ECM are usually produced byadjacent cells and secreted into the ECM via exocytosis. Once secreted,the ECM components often aggregate to form a complex network ofmacromolecules. In various embodiments, the chimeric protein of theinvention comprises a targeting moiety that recognizes a target (e.g.,an antigen or receptor or non-proteinaceous molecule) located on anycomponent of the ECM. Illustrative components of the ECM include,without limitation, the proteoglycans, the non-proteoglycanpolysaccharides, fibers, and other ECM proteins or ECM non-proteins,e.g. polysaccharides and/or lipids, or ECM associated molecules (e.g.proteins or non-proteins, e.g. polysaccharides, nucleic acids and/orlipids).

In some embodiments, the antigen recognition domain recognizes a target(e.g. antigen, receptor) on ECM proteoglycans. Proteoglycans areglycosylated proteins. The basic proteoglycan unit includes a coreprotein with one or more covalently attached glycosaminoglycan (GAG)chains. Proteoglycans have a net negative charge that attractspositively charged sodium ions (Na+), which attracts water molecules viaosmosis, keeping the ECM and resident cells hydrated. Proteoglycans mayalso help to trap and store growth factors within the ECM. Illustrativeproteoglycans that may be targeted by the chimeric proteins of theinvention include, but are not limited to, heparan sulfate, chondroitinsulfate, and keratan sulfate. In an embodiment, the targeting moietyrecognizes a target (e.g. antigen, receptor) on non-proteoglycanpolysaccharides such as hyaluronic acid.

In some embodiments, the antigen recognition domain recognizes a target(e.g. antigen, receptor) on ECM fibers. ECM fibers include collagenfibers and elastin fibers. In some embodiments, the targeting moietyrecognizes one or more epitopes on collagens or collagen fibers.Collagens are the most abundant proteins in the ECM. Collagens arepresent in the ECM as fibrillar proteins and provide structural supportto resident cells. In one or more embodiments, the targeting moietyrecognizes and binds to various types of collagens present within theECM including, without limitation, fibrillar collagens (types I, II,III, V, XI), facit collagens (types IX, XII, XIV), short chain collagens(types VIII, X), basement membrane collagens (type IV), and/or collagentypes VI, VII, or XIII. Elastin fibers provide elasticity to tissues,allowing them to stretch when needed and then return to their originalstate. In some embodiments, the target moiety recognizes one or moreepitopes on elastins or elastin fibers.

In some embodiments, the antigen recognition domain recognizes one ormore ECM proteins including, but not limited to, a tenascin, afibronectin, a fibrin, a laminin, or a nidogen/entactin.

In an embodiment, the antigen recognition domain recognizes and binds totenascin. The tenascin (TN) family of glycoproteins includes at leastfour members, tenascin-C, tenascin-R, tenascin-X, and tenascin W. Theprimary structures of tenascin proteins include several common motifsordered in the same consecutive sequence: amino-terminal heptad repeats,epidermal growth factor (EGF)-like repeats, fibronectin type III domainrepeats, and a carboxyl-terminal fibrinogen-like globular domain. Eachprotein member is associated with typical variations in the number andnature of EGF-like and fibronectin type III repeats. Isoform variantsalso exist particularly with respect to tenascin-C. Over 27 splicevariants and/or isoforms of tenascin-C are known. In a particularembodiment, the targeting moiety recognizes and binds to tenascin-CA1.Similarly, tenascin-R also has various splice variants and isoforms.Tenascin-R usually exists as dimers or trimers. Tenascin-X is thelargest member of the tenascin family and is known to exist as trimers.Tenascin-W exists as trimers. In some embodiments, the targeting moietyrecognizes one or more epitopes on a tenascin protein. In someembodiments, the targeting moiety recognizes the monomeric and/or thedimeric and/or the trimeric and/or the hexameric forms of a tenascinprotein.

In an embodiment, the antigen recognition domain recognizes and binds tofibronectin. Fibronectins are glycoproteins that connect cells withcollagen fibers in the ECM, allowing cells to move through the ECM. Uponbinding to integrins, fibronectins unfolds to form functional dimers. Insome embodiments, the targeting moiety recognizes the monomeric and/orthe dimeric forms of fibronectin. In some embodiments, the targetingmoiety recognizes one or more epitopes on fibronectin. In illustrativeembodiments, the targeting moiety recognizes fibronectin extracellulardomain A (EDA) or fibronectin extracellular domain B (EDB). Elevatedlevels of EDA are associated with various diseases and disordersincluding psoriasis, rheumatoid arthritis, diabetes, and cancer. In someembodiments, the targeting moiety recognizes fibronectin that containsthe EDA isoform and may be utilized to target the chimeric protein todiseased cells including cancer cells. In some embodiments, thetargeting moiety recognizes fibronectin that contains the EDB isoform.In various embodiments, such targeting moieties may be utilized totarget the chimeric protein to tumor cells including the tumorneovasculature.

In an embodiment, the antigen recognition domain recognizes and binds tofibrin. Fibrin is another protein substance often found in the matrixnetwork of the ECM. Fibrin is formed by the action of the proteasethrombin on fibrinogen which causes the fibrin to polymerize. In someembodiments, the targeting moiety recognizes one or more epitopes onfibrin. In some embodiments, the targeting moiety recognizes themonomeric as well as the polymerized forms of fibrin.

In an embodiment, the antigen recognition domain recognizes and binds tolaminin. Laminin is a major component of the basal lamina, which is aprotein network foundation for cells and organs. Laminins areheterotrimeric proteins that contain an α-chain, a β-chain, and aγ-chain. In some embodiments, the targeting moiety recognizes one ormore epitopes on laminin. In some embodiments, the targeting moietyrecognizes the monomeric, the dimeric as well as the trimeric forms oflaminin.

In an embodiment, the antigen recognition domain recognizes and binds toa nidogen or entactin. Nidogens/entactins are a family of highlyconserved, sulfated glycoproteins. They make up the major structuralcomponent of the basement membranes and function to link laminin andcollagen IV networks in basement membranes. Members of this familyinclude nidogen-1 and nidogen-2. In various embodiments, the targetingmoiety recognizes an epitope on nidogen-1 and/or nidogen-2.

In various embodiments, the antigen recognition domain recognizes anepitope present on any of the targets (e.g., ECM proteins) describedherein. In an embodiment, the antigen-recognition domain recognizes oneor more linear epitopes present on the protein. As used herein, a linearepitope refers to any continuous sequence of amino acids present on theprotein. In another embodiment, the antigen-recognition domainrecognizes one or more conformational epitopes present on the protein.As used herein, a conformation epitope refers to one or more sections ofamino acids (which may be discontinuous) which form a three-dimensionalsurface with features and/or shapes and/or tertiary structures capableof being recognized by an antigen recognition domain.

In various embodiments, the antigen recognition domain may bind to thefull-length and/or mature forms and/or isoforms and/or splice variantsand/or fragments and/or any other naturally occurring or syntheticanalogs, variants, or mutants of any of the targets (e.g., ECM proteins)described herein. In various embodiments, the antigen recognition domainmay bind to any forms of the proteins described herein, includingmonomeric, dimeric, trimeric, tetrameric, heterodimeric, multimeric andassociated forms. In various embodiments, the targeting moiety may bindto any post-translationally modified forms of the proteins describedherein, such as glycosylated and/or phosphorylated forms.

In various embodiments, the antigen recognition domain that recognizesextracellular molecules such as DNA. In some embodiments, the targetingmoiety comprises an antigen recognition domain that recognizes DNA. Inan embodiment, the DNA is shed into the extracellular space fromnecrotic or apoptotic tumor cells or other diseased cells.

In various embodiments, the antigen recognition domain that recognizesone or more non-cellular structures associated with atheroscleroticplaques. Two types of atherosclerotic plaques are known. The fibro-lipid(fibro-fatty) plaque is characterized by an accumulation of lipid-ladencells underneath the intima of the arteries. Beneath the endotheliumthere is a fibrous cap covering the atheromatous core of the plaque. Thecore includes lipid-laden cells (macrophages and smooth muscle cells)with elevated tissue cholesterol and cholesterol ester content, fibrin,proteoglycans, collagen, elastin, and cellular debris. In advancedplaques, the central core of the plaque usually contains extracellularcholesterol deposits (released from dead cells), which form areas ofcholesterol crystals with empty, needle-like clefts. At the periphery ofthe plaque are younger foamy cells and capillaries. A fibrous plaque isalso localized under the intima, within the wall of the artery resultingin thickening and expansion of the wall and, sometimes, spotty localizednarrowing of the lumen with some atrophy of the muscular layer. Thefibrous plaque contains collagen fibers (eosinophilic), precipitates ofcalcium (hematoxylinophilic) and lipid-laden cells. In some embodiments,the targeting moiety recognizes and binds to one or more of thenon-cellular components of these plaques such as the fibrin,proteoglycans, collagen, elastin, cellular debris, and calcium or othermineral deposits or precipitates. In some embodiments, the cellulardebris is a nucleic acid, e.g. DNA or RNA, released from dead cells.

In various embodiments, the antigen recognition domain that recognizesone or more non-cellular structures found in the brain plaquesassociated with neurodegenerative diseases. In some embodiments, thetargeting moiety recognizes and binds to one or more non-cellularstructures located in the amyloid plaques found in the brains ofpatients with Alzheimer's disease. For example, the targeting moiety mayrecognize and bind to the peptide amyloid beta, which is a majorcomponent of the amyloid plaques. In some embodiments, the targetingmoiety recognizes and binds to one or more non-cellular structureslocated in the brains plaques found in patients with Huntington'sdisease. In various embodiments, the targeting moiety recognizes andbinds to one or more non-cellular structures found in plaques associatedwith other neurodegenerative or musculoskeletal diseases such as Lewybody dementia and inclusion body myositis.

Targeting Moiety Formats

In various embodiments, the targeting moiety of the present chimericprotein is a protein-based agent capable of specific binding, such as anantibody or derivative thereof. For example, in various embodiments, thetargeting moiety of the present chimeric protein is an antibody (e.g. aclassic antibody or derivative thereof), single-domain antibody, arecombinant heavy-chain-only antibody (VHH), a single-chain antibody(scFv), a shark heavy-chain-only antibody (VNAR), a microprotein(cysteine knot protein, knottin), a DARPin; a Tetranectin; an Affibody;a Transbody; an Anticalin; an AdNectin; an Affilin; a Microbody; aDiabody, a peptide aptamer; an alterases; a plastic antibodies; aphylomer; a stradobodies; a maxibodies; an evibody; a fynomer, anarmadillo repeat protein, a Kunitz domain, an avimer, an atrimer, aprobody, an immunobody, a triomab, a troybody; a pepbody; a vaccibody, aUniBody; a DuoBody, a Fv, a Fab, a Fab′, a F(ab′)₂, a peptide mimeticmolecule, or a synthetic molecule, as described in U.S. Pat. Nos. orPatent Publication Nos. U.S. Pat. No. 7,417,130, US 2004/132094, U.S.Pat. No. 5,831,012, US 2004/023334, U.S. Pat. Nos. 7,250,297, 6,818,418,US 2004/209243, U.S. Pat. Nos. 7,838,629, 7,186,524, 6,004,746,5,475,096, US 2004/146938, US 2004/157209, U.S. Pat. Nos. 6,994,982,6,794,144, US 2010/239633, U.S. Pat. No. 7,803,907, US 2010/119446,and/or U.S. Pat. No. 7,166,697, the contents of which are herebyincorporated by reference in their entireties. See also, Storz MAbs.2011 May-Jun.; 3(3): 310-317.

In one embodiment, the target moiety comprises a single-domain antibody,such as VHH from, for example, an organism that produces VHH antibodysuch as a camelid, a shark, or a designed VHH. VHHs are antibody-derivedtherapeutic proteins that contain the unique structural and functionalproperties of naturally-occurring heavy-chain antibodies. VHH technologyis based on fully functional antibodies from camelids that lack lightchains. These heavy-chain antibodies contain a single variable domain(VHH) and two constant domains (CH2 and CH3). VHHs are commerciallyavailable under the trademark of NANOBODIES. In various embodiments, thetargeting moiety is a single-domain antibody (NANOBODY). In variousembodiments, the targeting moiety is a VHH. In an embodiment, thetargeting moiety is a humanized VHH. In another embodiment, thetargeting moiety is a camelized VHH.

In various embodiments, the present chimeric proteins comprise atargeting moiety as described herein.

In an illustrative embodiment, the targeting moiety of the presentchimeric protein may be in the context of chimeric protein thatcomprises a mutant soluble agent bearing mutations that attenuateactivity at a therapeutic receptor and substantially reduced or ablatedbinding at a second receptor.

Linkers

In some embodiments, the present chimeric protein comprises one or morelinkers.

In some embodiments vectors encoding the present chimeric proteinslinked as a single nucleotide sequence to any of the linkers describedherein are provided and may be used to prepare such chimeric proteins.

In some embodiments, the linker length allows for efficient binding ofthe targeting moiety and the modified soluble agent to their receptors.For instance, in some embodiments, the linker length allows forefficient binding of the targeting moiety and the modified soluble agentto receptors on the same cell.

In some embodiments the linker length is at least equal to the minimumdistance between the binding site of the targeting moiety and thereceptor for the modified soluble agent on the same cell. In someembodiments the linker length is at least twice, or three times, or fourtimes, or five times, or ten times the minimum distance between thebinding site of the targeting moiety and the modified soluble agent toreceptors on the same cell.

As described herein, the linker length allows for efficient binding ofthe targeting moiety and the modified soluble agent to receptors on thesame cell, the binding being sequential, e.g., targeting moiety/receptorbinding preceding modified soluble agent/receptor binding.

In various embodiments, a variety of linker sequences may be used. Invarious embodiments, the linker may be derived from naturally-occurringmulti-domain proteins or are empirical linkers as described, forexample, in Chichili et al., (2013), Protein Sci. 22(2):153-167, Chen etal., (2013), Adv Drug Deliv Rev. 65(10):1357-1369, the entire contentsof which are hereby incorporated by reference. In some embodiments, thelinker may be designed using linker designing databases and computerprograms such as those described in Chen et al., (2013), Adv Drug DelivRev. 65(10):1357-1369 and Crasto et al., (2000), Protein Eng.13(5):309-312, the entire contents of which are hereby incorporated byreference. In various embodiments, the linker may be functional. Forexample, without limitation, the linker may function to improve thefolding and/or stability, improve the expression, improve thepharmacokinetics, and/or improve the bioactivity of the present chimericprotein.

In some embodiments, the linker is a polypeptide. In some embodiments,the linker is less than about 100 amino acids long. For example, thelinker may be less than about 100, about 95, about 90, about 85, about80, about 75, about 70, about 65, about 60, about 55, about 50, about45, about 40, about 35, about 30, about 25, about 20, about 19, about18, about 17, about 16, about 15, about 14, about 13, about 12, about11, about 10, about 9, about 8, about 7, about 6, about 5, about 4,about 3, or about 2 amino acids long. In some embodiments, the linker isflexible. In another embodiment, the linker is rigid.

In various embodiments, the linker is substantially comprised of glycineand serine residues (e.g. about 30%, or about 40%, or about 50%, orabout 60%, or about 70%, or about 80%, or about 90%, or about 95%, orabout 97% glycines and serines). For example, in some embodiments, thelinker is (Gly₄Ser)_(n), where n is from about 1 to about 8, e.g. 1, 2,3, 4, 5, 6, 7, or 8. In an embodiment, the linker sequence isGGSGGSGGGGSGGGGS (SEQ ID NO: 241). Additional illustrative linkersinclude, but are not limited to, linkers having the sequence LE, GGGGS(SEQ ID NO: 242), (GGGGS)_(n) (n=1-4)(SEQ ID NO: 243), (Gly)₈ (SEQ IDNO: 244), (Gly)₆ (SEQ ID NO: 245), (EAAAK)_(n) (n=1-3)(SEQ ID NO: 246),A(EAAAK)_(n)A (n=2-5)(SEQ ID NO: 247), AEAAAKEAAAKA (SEQ ID NO: 248),A(EAAAK)₄ALEA(EAAAK)₄A (SEQ ID NO: 249), PAPAP (SEQ ID NO: 250),KESGSVSSEQLAQFRSLD (SEQ ID NO: 251), EGKSSGSGSESKST (SEQ ID NO: 252),GSAGSAAGSGEF (SEQ ID NO: 253), and (XP)_(n), with X designating anyamino acid, e.g., Ala, Lys, or Glu. In various embodiments, the linkeris GGS. In an embodiment, the linker is 20×GGS (SEQ ID NO: 254).

In some embodiments, the linker is a hinge region of an antibody (e.g.,of IgG, IgA, IgD, and IgE, inclusive of subclasses (e.g. IgG1, IgG2,IgG3, and IgG4, and IgA1 and IgA2)). In various embodiments, the linkeris a hinge region of an antibody (e.g., of IgG, IgA, IgD, and IgE,inclusive of subclasses (e.g. IgG1, IgG2, IgG3, and IgG4, and IgA1 andIgA2)). The hinge region, found in IgG, IgA, IgD, and IgE classantibodies, acts as a flexible spacer, allowing the Fab portion to movefreely in space. In contrast to the constant regions, the hinge domainsare structurally diverse, varying in both sequence and length amongimmunoglobulin classes and subclasses. For example, the length andflexibility of the hinge region varies among the IgG subclasses. Thehinge region of IgG1 encompasses amino acids 216-231 and, because it isfreely flexible, the Fab fragments can rotate about their axes ofsymmetry and move within a sphere centered at the first of twointer-heavy chain disulfide bridges. IgG2 has a shorter hinge than IgG1,with 12 amino acid residues and four disulfide bridges. The hinge regionof IgG2 lacks a glycine residue, is relatively short, and contains arigid poly-proline double helix, stabilized by extra inter-heavy chaindisulfide bridges. These properties restrict the flexibility of the IgG2molecule. IgG3 differs from the other subclasses by its unique extendedhinge region (about four times as long as the IgG1 hinge), containing 62amino acids (including 21 prolines and 11 cysteines), forming aninflexible poly-proline double helix. In IgG3, the Fab fragments arerelatively far away from the Fc fragment, giving the molecule a greaterflexibility. The elongated hinge in IgG3 is also responsible for itshigher molecular weight compared to the other subclasses. The hingeregion of IgG4 is shorter than that of IgG1 and its flexibility isintermediate between that of IgG1 and IgG2. The flexibility of the hingeregions reportedly decreases in the order IgG3>IgG1>IgG4>IgG2.

According to crystallographic studies, the immunoglobulin hinge regioncan be further subdivided functionally into three regions: the upperhinge region, the core region, and the lower hinge region. See Shin etal., 1992 Immunological Reviews 130:87. The upper hinge region includesamino acids from the carboxyl end of C_(H1) to the first residue in thehinge that restricts motion, generally the first cysteine residue thatforms an interchain disulfide bond between the two heavy chains. Thelength of the upper hinge region correlates with the segmentalflexibility of the antibody. The core hinge region contains theinter-heavy chain disulfide bridges, and the lower hinge region joinsthe amino terminal end of the C_(H2) domain and includes residues inC_(H2). Id. The core hinge region of wild-type human IgG1 contains thesequence Cys-Pro-Pro-Cys which, when dimerized by disulfide bondformation, results in a cyclic octapeptide believed to act as a pivot,thus conferring flexibility. In various embodiments, the present linkercomprises, one, or two, or three of the upper hinge region, the coreregion, and the lower hinge region of any antibody (e.g., of IgG, IgA,IgD, and IgE, inclusive of subclasses (e.g. IgG1, IgG2, IgG3, and IgG4,and IgA1 and IgA2)). The hinge region may also contain one or moreglycosylation sites, which include a number of structurally distincttypes of sites for carbohydrate attachment. For example, IgA1 containsfive glycosylation sites within a 17-amino-acid segment of the hingeregion, conferring resistance of the hinge region polypeptide tointestinal proteases, considered an advantageous property for asecretory immunoglobulin. In various embodiments, the linker of thepresent invention comprises one or more glycosylation sites. In variousembodiments, the linker is a hinge-CH2-CH3 domain of a human IgG4antibody.

If desired, the present chimeric protein can be linked to an antibody Fcregion, comprising one or both of C_(H)2 and C_(H)3 domains, andoptionally a hinge region. For example, vectors encoding the presentchimeric proteins linked as a single nucleotide sequence to an Fc regioncan be used to prepare such polypeptides.

In some embodiments, the linker is a synthetic linker such as PEG.

In various embodiments, the linker may be functional. For example,without limitation, the linker may function to improve the foldingand/or stability, improve the expression, improve the pharmacokinetics,and/or improve the bioactivity of the present chimeric protein. Inanother example, the linker may function to target the chimeric proteinto a particular cell type or location. In various embodiments, thepresent chimeric proteins may by conjugated and/or fused with anotheragent to extend half-life or otherwise improve pharmacodynamic andpharmacokinetic properties. In some embodiments, the chimeric proteinsmay be fused or conjugated with one or more of PEG, XTEN (e.g., asrPEG), polysialic acid (POLYXEN), albumin (e.g., human serum albumin orHAS), elastin-like protein (ELP), PAS, HAP, GLK, CTP, transferrin, andthe like. In some embodiments, the chimeric protein may be fused orconjugated with an antibody or an antibody fragment such as an Fcfragment. For example, the chimeric protein may be fused to either theN-terminus or the C-terminus of the Fc domain of human immunoglobulin(Ig) G. In various embodiments, each of the individual chimeric proteinsis fused to one or more of the agents described in BioDrugs (2015)29:215-239, the entire contents of which are hereby incorporated byreference.

Production of Chimeric Proteins

Methods for producing the chimeric proteins of the invention aredescribed herein. For example, DNA sequences encoding the chimericproteins of the invention (e.g., DNA sequences encoding the modifiedsoluble agent and the targeting moiety and the linker) can be chemicallysynthesized using methods known in the art. Synthetic DNA sequences canbe ligated to other appropriate nucleotide sequences, including, e.g.,expression control sequences, to produce gene expression constructsencoding the desired chimeric proteins. Accordingly, in variousembodiments, the present invention provides for isolated nucleic acidscomprising a nucleotide sequence encoding the chimeric protein of theinvention.

Nucleic acids encoding the chimeric protein of the invention can beincorporated (ligated) into expression vectors, which can be introducedinto host cells through transfection, transformation, or transductiontechniques. For example, nucleic acids encoding the chimeric protein ofthe invention can be introduced into host cells by retroviraltransduction. Illustrative host cells are E. coli cells, Chinese hamsterovary (CHO) cells, human embryonic kidney 293 (HEK 293) cells, HeLacells, baby hamster kidney (BHK) cells, monkey kidney cells (COS), humanhepatocellular carcinoma cells (e.g., Hep G2), and myeloma cells.Transformed host cells can be grown under conditions that permit thehost cells to express the genes that encode the chimeric protein of theinvention. Accordingly, in various embodiments, the present inventionprovides expression vectors comprising nucleic acids that encode thechimeric protein of the invention. In various embodiments, the presentinvention additional provides host cells comprising such expressionvectors.

Specific expression and purification conditions will vary depending uponthe expression system employed. For example, if a gene is to beexpressed in E. coli, it is first cloned into an expression vector bypositioning the engineered gene downstream from a suitable bacterialpromoter, e.g., Trp or Tac, and a prokaryotic signal sequence. Inanother example, if the engineered gene is to be expressed in eukaryotichost cells, e.g., CHO cells, it is first inserted into an expressionvector containing for example, a suitable eukaryotic promoter, asecretion signal, enhancers, and various introns. The gene construct canbe introduced into the host cells using transfection, transformation, ortransduction techniques.

The chimeric protein of the invention can be produced by growing a hostcell transfected with an expression vector encoding the chimeric proteinunder conditions that permit expression of the protein. Followingexpression, the protein can be harvested and purified using techniqueswell known in the art, e.g., affinity tags such asglutathione-S-transferase (GST) and histidine tags or by chromatography.

Accordingly, in various embodiments, the present invention provides fora nucleic acid encoding a chimeric protein of the present invention. Invarious embodiments, the present invention provides for a host cellcomprising a nucleic acid encoding a chimeric protein of the presentinvention.

Pharmaceutically Acceptable Salts and Excipients

The chimeric proteins described herein can possess a sufficiently basicfunctional group, which can react with an inorganic or organic acid, ora carboxyl group, which can react with an inorganic or organic base, toform a pharmaceutically acceptable salt. A pharmaceutically acceptableacid addition salt is formed from a pharmaceutically acceptable acid, asis well known in the art. Such salts include the pharmaceuticallyacceptable salts listed in, for example, Journal of PharmaceuticalScience, 66, 2-19 (1977) and The Handbook of Pharmaceutical Salts;Properties, Selection, and Use. P. H. Stahl and C. G. Wermuth (eds.),Verlag, Zurich (Switzerland) 2002, which are hereby incorporated byreference in their entirety.

Pharmaceutically acceptable salts include, by way of non-limitingexample, sulfate, citrate, acetate, oxalate, chloride, bromide, iodide,nitrate, bisulfate, phosphate, acid phosphate, isonicotinate, lactate,salicylate, acid citrate, tartrate, oleate, tannate, pantothenate,bitartrate, ascorbate, succinate, maleate, gentisinate, fumarate,gluconate, glucaronate, saccharate, formate, benzoate, glutamate,methanesulfonate, ethanesulfonate, benzenesulfonate, p-toluenesulfonate,camphorsulfonate, pamoate, phenylacetate, trifluoroacetate, acrylate,chlorobenzoate, dinitrobenzoate, hydroxybenzoate, methoxybenzoate,methylbenzoate, o-acetoxybenzoate, naphthalene-2-benzoate, isobutyrate,phenylbutyrate, α-hydroxybutyrate, butyne-1,4-dicarboxylate,hexyne-1,4-dicarboxylate, caprate, caprylate, cinnamate, glycollate,heptanoate, hippurate, malate, hydroxymaleate, malonate, mandelate,mesylate, nicotinate, phthalate, teraphthalate, propiolate, propionate,phenylpropionate, sebacate, suberate, p-bromobenzenesulfonate,chlorobenzenesulfonate, ethylsulfonate, 2-hydroxyethylsulfonate,methylsulfonate, naphthalene-1-sulfonate, naphthalene-2-sulfonate,naphthalene-1,5-sulfonate, xylenesulfonate, and tartarate salts.

The term “pharmaceutically acceptable salt” also refers to a salt of thecompositions of the present invention having an acidic functional group,such as a carboxylic acid functional group, and a base. Suitable basesinclude, but are not limited to, hydroxides of alkali metals such assodium, potassium, and lithium; hydroxides of alkaline earth metal suchas calcium and magnesium; hydroxides of other metals, such as aluminumand zinc; ammonia, and organic amines, such as unsubstituted orhydroxy-substituted mono-, di-, or tri-alkylamines, dicyclohexylamine;tributyl amine; pyridine; N-methyl, N-ethylamine; diethylamine;triethylamine; mono-, bis-, or tris-(2-OH-lower alkylamines), such asmono-; bis-, or tris-(2-hydroxyethyl)amine, 2-hydroxy-tert-butylamine,or tris-(hydroxymethyl)methylamine, N,N-di-lower alkyl-N-(hydroxyl-loweralkyl)-amines, such as N,N-dimethyl-N-(2-hydroxyethyl)amine ortri-(2-hydroxyethyl)amine; N-methyl-D-glucamine; and amino acids such asarginine, lysine, and the like.

In some embodiments, the compositions described herein are in the formof a pharmaceutically acceptable salt.

Pharmaceutical Compositions and Formulations

In various embodiments, the present invention pertains to pharmaceuticalcompositions comprising the chimeric proteins described herein and apharmaceutically acceptable carrier or excipient. Any pharmaceuticalcompositions described herein can be administered to a subject as acomponent of a composition that comprises a pharmaceutically acceptablecarrier or vehicle. Such compositions can optionally comprise a suitableamount of a pharmaceutically acceptable excipient so as to provide theform for proper administration.

In various embodiments, pharmaceutical excipients can be liquids, suchas water and oils, including those of petroleum, animal, vegetable, orsynthetic origin, such as peanut oil, soybean oil, mineral oil, sesameoil and the like. The pharmaceutical excipients can be, for example,saline, gum acacia, gelatin, starch paste, talc, keratin, colloidalsilica, urea and the like. In addition, auxiliary, stabilizing,thickening, lubricating, and coloring agents can be used. In oneembodiment, the pharmaceutically acceptable excipients are sterile whenadministered to a subject. Water is a useful excipient when any agentdescribed herein is administered intravenously. Saline solutions andaqueous dextrose and glycerol solutions can also be employed as liquidexcipients, specifically for injectable solutions. Suitablepharmaceutical excipients also include starch, glucose, lactose,sucrose, gelatin, malt, rice, flour, chalk, silica gel, sodium stearate,glycerol monostearate, talc, sodium chloride, dried skim milk, glycerol,propylene, glycol, water, ethanol and the like. Any agent describedherein, if desired, can also comprise minor amounts of wetting oremulsifying agents, or pH buffering agents. Other examples of suitablepharmaceutical excipients are described in Remington's PharmaceuticalSciences 1447-1676 (Alfonso R. Gennaro eds., 19th ed. 1995),incorporated herein by reference.

The present invention includes the described pharmaceutical compositions(and/or additional therapeutic agents) in various formulations. Anyinventive pharmaceutical composition (and/or additional therapeuticagents) described herein can take the form of solutions, suspensions,emulsion, drops, tablets, pills, pellets, capsules, capsules containingliquids, gelatin capsules, powders, sustained-release formulations,suppositories, emulsions, aerosols, sprays, suspensions, lyophilizedpowder, frozen suspension, desiccated powder, or any other form suitablefor use. In one embodiment, the composition is in the form of a capsule.In another embodiment, the composition is in the form of a tablet. Inyet another embodiment, the pharmaceutical composition is formulated inthe form of a soft-gel capsule. In a further embodiment, thepharmaceutical composition is formulated in the form of a gelatincapsule. In yet another embodiment, the pharmaceutical composition isformulated as a liquid.

Where necessary, the inventive pharmaceutical compositions (and/oradditional agents) can also include a solubilizing agent. Also, theagents can be delivered with a suitable vehicle or delivery device asknown in the art. Combination therapies outlined herein can beco-delivered in a single delivery vehicle or delivery device.

The formulations comprising the inventive pharmaceutical compositions(and/or additional agents) of the present invention may conveniently bepresented in unit dosage forms and may be prepared by any of the methodswell known in the art of pharmacy. Such methods generally include thestep of bringing the therapeutic agents into association with a carrier,which constitutes one or more accessory ingredients. Typically, theformulations are prepared by uniformly and intimately bringing thetherapeutic agent into association with a liquid carrier, a finelydivided solid carrier, or both, and then, if necessary, shaping theproduct into dosage forms of the desired formulation (e.g., wet or drygranulation, powder blends, etc., followed by tableting usingconventional methods known in the art).

In various embodiments, any pharmaceutical compositions (and/oradditional agents) described herein is formulated in accordance withroutine procedures as a composition adapted for a mode of administrationdescribed herein.

Routes of administration include, for example: oral, intradermal,intramuscular, intraperitoneal, intravenous, subcutaneous, intranasal,epidural, sublingual, intranasal, intracerebral, intravaginal,transdermal, rectally, by inhalation, or topically. Administration canbe local or systemic. In some embodiments, the administering is effectedorally. In another embodiment, the administration is by parenteralinjection. The mode of administration can be left to the discretion ofthe practitioner, and depends in-part upon the site of the medicalcondition. In most instances, administration results in the release ofany agent described herein into the bloodstream.

In one embodiment, the chimeric protein described herein is formulatedin accordance with routine procedures as a composition adapted for oraladministration. Compositions for oral delivery can be in the form oftablets, lozenges, aqueous or oily suspensions, granules, powders,emulsions, capsules, syrups, or elixirs, for example. Orallyadministered compositions can comprise one or more agents, for example,sweetening agents such as fructose, aspartame or saccharin; flavoringagents such as peppermint, oil of wintergreen, or cherry; coloringagents; and preserving agents, to provide a pharmaceutically palatablepreparation. Moreover, where in tablet or pill form, the compositionscan be coated to delay disintegration and absorption in thegastrointestinal tract thereby providing a sustained action over anextended period of time. Selectively permeable membranes surrounding anosmotically active driving any chimeric proteins described herein arealso suitable for orally administered compositions. In these latterplatforms, fluid from the environment surrounding the capsule is imbibedby the driving compound, which swells to displace the agent or agentcomposition through an aperture. These delivery platforms can provide anessentially zero order delivery profile as opposed to the spikedprofiles of immediate release formulations. A time-delay material suchas glycerol monostearate or glycerol stearate can also be useful. Oralcompositions can include standard excipients such as mannitol, lactose,starch, magnesium stearate, sodium saccharin, cellulose, and magnesiumcarbonate. In one embodiment, the excipients are of pharmaceuticalgrade. Suspensions, in addition to the active compounds, may containsuspending agents such as, for example, ethoxylated isostearyl alcohols,polyoxyethylene sorbitol and sorbitan esters, microcrystallinecellulose, aluminum metahydroxide, bentonite, agar-agar, tragacanth,etc., and mixtures thereof.

Dosage forms suitable for parenteral administration (e.g. intravenous,intramuscular, intraperitoneal, subcutaneous and intra-articularinjection and infusion) include, for example, solutions, suspensions,dispersions, emulsions, and the like. They may also be manufactured inthe form of sterile solid compositions (e.g. lyophilized composition),which can be dissolved or suspended in sterile injectable mediumimmediately before use. They may contain, for example, suspending ordispersing agents known in the art. Formulation components suitable forparenteral administration include a sterile diluent such as water forinjection, saline solution, fixed oils, polyethylene glycols, glycerine,propylene glycol or other synthetic solvents; antibacterial agents suchas benzyl alcohol or methyl paraben; antioxidants such as ascorbic acidor sodium bisulfite; chelating agents such as EDTA; buffers such asacetates, citrates or phosphates; and agents for the adjustment oftonicity such as sodium chloride or dextrose.

For intravenous administration, suitable carriers include physiologicalsaline, bacteriostatic water, Cremophor ELTM (BASF, Parsippany, N.J.) orphosphate buffered saline (PBS). The carrier should be stable under theconditions of manufacture and storage, and should be preserved againstmicroorganisms. The carrier can be a solvent or dispersion mediumcontaining, for example, water, ethanol, polyol (for example, glycerol,propylene glycol, and liquid polyethylene glycol), and suitable mixturesthereof.

The compositions provided herein, alone or in combination with othersuitable components, can be made into aerosol formulations (i.e.,“nebulized”) to be administered via inhalation. Aerosol formulations canbe placed into pressurized acceptable propellants, such asdichlorodifluoromethane, propane, nitrogen, and the like.

Any inventive pharmaceutical compositions (and/or additional agents)described herein can be administered by controlled-release orsustained-release means or by delivery devices that are well known tothose of ordinary skill in the art. Examples include, but are notlimited to, those described in U.S. Pat. Nos. 3,845,770; 3,916,899;3,536,809; 3,598,123; 4,008,719; 5,674,533; 5,059,595; 5,591,767;5,120,548; 5,073,543; 5,639,476; 5,354,556; and 5,733,556, each of whichis incorporated herein by reference in its entirety. Such dosage formscan be useful for providing controlled- or sustained-release of one ormore active ingredients using, for example, hydropropyl cellulose,hydropropylmethyl cellulose, polyvinylpyrrolidone, other polymermatrices, gels, permeable membranes, osmotic systems, multilayercoatings, microparticles, liposomes, microspheres, or a combinationthereof to provide the desired release profile in varying proportions.Suitable controlled- or sustained-release formulations known to thoseskilled in the art, including those described herein, can be readilyselected for use with the active ingredients of the agents describedherein. The invention thus provides single unit dosage forms suitablefor oral administration such as, but not limited to, tablets, capsules,gelcaps, and caplets that are adapted for controlled- orsustained-release.

Controlled- or sustained-release of an active ingredient can bestimulated by various conditions, including but not limited to, changesin pH, changes in temperature, stimulation by an appropriate wavelengthof light, concentration or availability of enzymes, concentration oravailability of water, or other physiological conditions or compounds.

In another embodiment, a controlled-release system can be placed inproximity of the target area to be treated, thus requiring only afraction of the systemic dose (see, e.g., Goodson, in MedicalApplications of Controlled Release, supra, vol. 2, pp. 115-138 (1984)).Other controlled-release systems discussed in the review by Langer,1990, Science 249:1527-1533) may be used.

Pharmaceutical formulations preferably are sterile. Sterilization can beaccomplished, for example, by filtration through sterile filtrationmembranes. Where the composition is lyophilized, filter sterilizationcan be conducted prior to or following lyophilization andreconstitution.

Administration and Dosage

It will be appreciated that the actual dose of the chimeric protein tobe administered according to the present invention will vary accordingto the particular dosage form, and the mode of administration. Manyfactors that may modify the action of the chimeric protein (e.g., bodyweight, gender, diet, time of administration, route of administration,rate of excretion, condition of the subject, drug combinations, geneticdisposition and reaction sensitivities) can be taken into account bythose skilled in the art. Administration can be carried out continuouslyor in one or more discrete doses within the maximum tolerated dose.Optimal administration rates for a given set of conditions can beascertained by those skilled in the art using conventional dosageadministration tests.

In some embodiments, a suitable dosage of the chimeric protein is in arange of about 0.01 mg/kg to about 10 g/kg of body weight of thesubject, about 0.01 mg/kg to about 1 g/kg of body weight of the subject,about 0.01 mg/kg to about 100 mg/kg of body weight of the subject, about0.01 mg/kg to about 10 mg/kg of body weight of the subject, for example,about 0.01 mg/kg, about 0.02 mg/kg, about 0.03 mg/kg, about 0.04 mg/kg,about 0.05 mg/kg, about 0.06 mg/kg, about 0.07 mg/kg, about 0.08 mg/kg,about 0.09 mg/kg, about 0.1 mg/kg, about 0.2 mg/kg, about 0.3 mg/kg,about 0.4 mg/kg, about 0.5 mg/kg, about 0.6 mg/kg, about 0.7 mg/kg,about 0.8 mg/kg, about 0.9 mg/kg, about 1 mg/kg, about 1.1 mg/kg, about1.2 mg/kg, about 1.3 mg/kg, about 1.4 mg/kg, about 1.5 mg/kg, about 1.6mg/kg, about 1.7 mg/kg, about 1.8 mg/kg, 1.9 mg/kg, about 2 mg/kg, about3 mg/kg, about 4 mg/kg, about 5 mg/kg, about 6 mg/kg, about 7 mg/kg,about 8 mg/kg, about 9 mg/kg, about 10 mg/kg body weight, about 100mg/kg body weight, about 1 g/kg of body weight, about 10 g/kg of bodyweight, inclusive of all values and ranges therebetween.

Individual doses of the chimeric protein can be administered in unitdosage forms (e.g., tablets or capsules) containing, for example, fromabout 0.01 mg to about 100 g, from about 0.01 mg to about 75 g, fromabout 0.01 mg to about 50 g, from about 0.01 mg to about 25 g, about0.01 mg to about 10 g, about 0.01 mg to about 7.5 g, about 0.01 mg toabout 5 g, about 0.01 mg to about 2.5 g, about 0.01 mg to about 1 g,about 0.01 mg to about 100 mg, from about 0.1 mg to about 100 mg, fromabout 0.1 mg to about 90 mg, from about 0.1 mg to about 80 mg, fromabout 0.1 mg to about 70 mg, from about 0.1 mg to about 60 mg, fromabout 0.1 mg to about 50 mg, from about 0.1 mg to about 40 mg activeingredient, from about 0.1 mg to about 30 mg, from about 0.1 mg to about20 mg, from about 0.1 mg to about 10 mg, from about 0.1 mg to about 5mg, from about 0.1 mg to about 3 mg, from about 0.1 mg to about 1 mg perunit dosage form, or from about 5 mg to about 80 mg per unit dosageform. For example, a unit dosage form can be about 0.01 mg, about 0.02mg, about 0.03 mg, about 0.04 mg, about 0.05 mg, about 0.06 mg, about0.07 mg, about 0.08 mg, about 0.09 mg, about 0.1 mg, about 0.2 mg, about0.3 mg, about 0.4 mg, about 0.5 mg, about 0.6 mg, about 0.7 mg, about0.8 mg, about 0.9 mg, about 1 mg, about 2 mg, about 3 mg, about 4 mg,about 5 mg, about 6 mg, about 7 mg, about 8 mg, about 9 mg about 10 mg,about 15 mg, about 20 mg, about 25 mg, about 30 mg, about 35 mg, about40 mg, about 45 mg, about 50 mg, about 55 mg, about 60 mg, about 65 mg,about 70 mg, about 75 mg, about 80 mg, about 85 mg, about 90 mg, about95 mg, about 100 mg, about 200 mg, about 500 mg, about 1 g, about 2.5 g,about 5 g, about 10 g, about 25 g, about 50 g, about 75 g, about 100 g,inclusive of all values and ranges therebetween.

In one embodiment, the chimeric protein is administered at an amount offrom about 0.01 mg to about 100 g daily, from about 0.01 mg to about 75g daily, from about 0.01 mg to about 50 g daily, from about 0.01 mg toabout 25 g daily, from about 0.01 mg to about 10 g daily, from about0.01 mg to about 7.5 g daily, from about 0.01 mg to about 5 g daily,from about 0.01 mg to about 2.5 g daily, from about 0.01 mg to about 1 gdaily, from about 0.01 mg to about 100 mg daily, from about 0.1 mg toabout 100 mg daily, from about 0.1 mg to about 95 mg daily, from about0.1 mg to about 90 mg daily, from about 0.1 mg to about 85 mg daily,from about 0.1 mg to about 80 mg daily, from about 0.1 mg to about 75 mgdaily, from about 0.1 mg to about 70 mg daily, from about 0.1 mg toabout 65 mg daily, from about 0.1 mg to about 60 mg daily, from about0.1 mg to about 55 mg daily, from about 0.1 mg to about 50 mg daily,from about 0.1 mg to about 45 mg daily, from about 0.1 mg to about 40 mgdaily, from about 0.1 mg to about 35 mg daily, from about 0.1 mg toabout 30 mg daily, from about 0.1 mg to about 25 mg daily, from about0.1 mg to about 20 mg daily, from about 0.1 mg to about 15 mg daily,from about 0.1 mg to about 10 mg daily, from about 0.1 mg to about 5 mgdaily, from about 0.1 mg to about 3 mg daily, from about 0.1 mg to about1 mg daily, or from about 5 mg to about 80 mg daily. In variousembodiments, the chimeric protein is administered at a daily dose ofabout 0.01 mg, about 0.02 mg, about 0.03 mg, about 0.04 mg, about 0.05mg, about 0.06 mg, about 0.07 mg, about 0.08 mg, about 0.09 mg, about0.1 mg, about 0.2 mg, about 0.3 mg, about 0.4 mg, about 0.5 mg, about0.6 mg, about 0.7 mg, about 0.8 mg, about 0.9 mg, about 1 mg, about 2mg, about 3 mg, about 4 mg, about 5 mg, about 6 mg, about 7 mg, about 8mg, about 9 mg about 10 mg, about 15 mg, about 20 mg, about 25 mg, about30 mg, about 35 mg, about 40 mg, about 45 mg, about 50 mg, about 55 mg,about 60 mg, about 65 mg, about 70 mg, about 75 mg, about 80 mg, about85 mg, about 90 mg, about 95 mg, about 100 mg, about 200 mg, about 500mg, about 1 g, about 2.5 g, about 5 g, about 7.5 g, about 10 g, about 25g, about 50 g, about 75 g, about 100 g, inclusive of all values andranges therebetween.

In accordance with certain embodiments of the invention, thepharmaceutical composition comprising the chimeric protein may beadministered, for example, more than once daily (e.g., about two times,about three times, about four times, about five times, about six times,about seven times, about eight times, about nine times, or about tentimes daily), about once per day, about every other day, about everythird day, about once a week, about once every two weeks, about onceevery month, about once every two months, about once every three months,about once every six months, or about once every year.

Combination Therapy and Additional Therapeutic Agents

In various embodiments, the pharmaceutical composition of the presentinvention is co-administered in conjunction with additional therapeuticagent(s). Co-administration can be simultaneous or sequential.

In one embodiment, the additional therapeutic agent and the chimericprotein of the present invention are administered to a subjectsimultaneously. The term “simultaneously” as used herein, means that theadditional therapeutic agent and the chimeric protein are administeredwith a time separation of no more than about 60 minutes, such as no morethan about 30 minutes, no more than about 20 minutes, no more than about10 minutes, no more than about 5 minutes, or no more than about 1minute. Administration of the additional therapeutic agent and thechimeric protein can be by simultaneous administration of a singleformulation (e.g., a formulation comprising the additional therapeuticagent and the chimeric protein) or of separate formulations (e.g., afirst formulation including the additional therapeutic agent and asecond formulation including the chimeric protein).

Co-administration does not require the therapeutic agents to beadministered simultaneously, if the timing of their administration issuch that the pharmacological activities of the additional therapeuticagent and the chimeric protein overlap in time, thereby exerting acombined therapeutic effect. For example, the additional therapeuticagent and the chimeric protein can be administered sequentially. Theterm “sequentially” as used herein means that the additional therapeuticagent and the chimeric protein are administered with a time separationof more than about 60 minutes. For example, the time between thesequential administration of the additional therapeutic agent and thechimeric protein can be more than about 60 minutes, more than about 2hours, more than about 5 hours, more than about 10 hours, more thanabout 1 day, more than about 2 days, more than about 3 days, or morethan about 1 week apart. The optimal administration times will depend onthe rates of metabolism, excretion, and/or the pharmacodynamic activityof the additional therapeutic agent and the chimeric protein beingadministered. Either the additional therapeutic agent or the chimericprotein cell may be administered first.

Co-administration also does not require the therapeutic agents to beadministered to the subject by the same route of administration. Rather,each therapeutic agent can be administered by any appropriate route, forexample, parenterally or non-parenterally.

In some embodiments, the chimeric protein described herein actssynergistically when co-administered with another therapeutic agent. Insuch embodiments, the chimeric protein and the additional therapeuticagent may be administered at doses that are lower than the dosesemployed when the agents are used in the context of monotherapy.

In some embodiments, the present invention pertains to chemotherapeuticagents as additional therapeutic agents. Examples of chemotherapeuticagents include, but are not limited to, alkylating agents such asthiotepa and CYTOXAN cyclosphosphamide; alkyl sulfonates such asbusulfan, improsulfan and piposulfan; aziridines such as benzodopa,carboquone, meturedopa, and uredopa; ethylenimines and methylamelaminesincluding altretamine, triethylenemelamine, trietylenephosphoramide,triethiylenethiophosphoramide and trimethylolomelamine; acetogenins(e.g., bullatacin and bullatacinone); a camptothecin (including thesynthetic analogue topotecan); bryostatin; cally statin; CC-1065(including its adozelesin, carzelesin and bizelesin syntheticanalogues); cryptophycins (e.g., cryptophycin 1 and cryptophycin 8);dolastatin; duocarmycin (including the synthetic analogues, KW-2189 andCB 1-TM1); eleutherobin; pancratistatin; a sarcodictyin; spongistatin;nitrogen mustards such as chlorambucil, chlornaphazine,cholophosphamide, estramustine, ifosfamide, mechlorethamine,mechlorethamine oxide hydrochloride, melphalan, novembichin,phenesterine, prednimustine, trofosfamide, uracil mustard; nitrosureassuch as carmustine, chlorozotocin, fotemustine, lomustine, nimustine,and ranimnustine; antibiotics such as the enediyne antibiotics (e.g.,calicheamicin, especially calicheamicin gammall and calicheamicinomegall (see, e.g., Agnew, Chem. Intl. Ed. Engl., 33: 183-186 (1994));dynemicin, including dynemicin A; bisphosphonates, such as clodronate;an esperamicin; as well as neocarzinostatin chromophore and relatedchromoprotein enediyne antibiotic chromophores), adacinomysins,actinomycin, authramycin, azaserine, bleomycins, cactinomycin,carabicin, caminomycin, carzinophilin, chromomycinis, dactinomycin,daunorubicin, detorubicin, 6-diazo-5-oxo-L-norleucine, ADRIAMYCINdoxorubicin (including morpholino-doxorubicin,cyanomorpholino-doxorubicin, 2-pyrrolino-doxorubicin and deoxydoxorubicin), epirubicin, esorubicin, idarubicin, marcellomycin,mitomycins such as mitomycin C, mycophenolic acid, nogalamycin,olivomycins, peplomycin, potfiromycin, puromycin, quelamycin,rodorubicin, streptonigrin, streptozocin, tubercidin, ubenimex,zinostatin, zorubicin; anti-metabolites such as methotrexate and5-fluorouracil (5-FU); folic acid analogues such as denopterin,methotrexate, pteropterin, trimetrexate; purine analogs such asfludarabine, 6-mercaptopurine, thiamiprine, thioguanine; pyrimidineanalogs such as ancitabine, azacitidine, 6-azauridine, carmofur,cytarabine, dideoxyuridine, doxifluridine, enocitabine, floxuridine;androgens such as calusterone, dromostanolone propionate, epitiostanol,mepitiostane, testolactone; anti-adrenals such as minoglutethimide,mitotane, trilostane; folic acid replenisher such as frolinic acid;aceglatone; aldophosphamide glycoside; aminolevulinic acid; eniluracil;amsacrine; bestrabucil; bisantrene; edatraxate; demecolcine; diaziquone;elformithine; elliptinium acetate; an epothilone; etoglucid; galliumnitrate; hydroxyurea; lentinan; lonidainine; maytansinoids such asmaytansine and ansamitocins; mitoguazone; mitoxantrone; mopidanmol;nitraerine; pentostatin; phenamet; pirarubicin; losoxantrone;podophyllinic acid; 2-ethylhydrazide; procarbazine; PSK polysaccharidecomplex (JHS Natural Products, Eugene, Oreg.); razoxane; rhizoxin;sizofuran; spirogermanium; tenuazonic acid; triaziquone;2,2′,2″-trichlorotriethylamine; trichothecenes (e.g., T-2 toxin,verracurin A, roridin A and anguidine); urethan; vindesine; dacarbazine;mannomustine; mitobronitol; mitolactol; pipobroman; gacytosine;arabinoside (“Ara-C”); cyclophosphamide; thiotepa; taxoids, e.g., TAXOLpaclitaxel (Bristol-Myers Squibb Oncology, Princeton, N.J.), ABRAXANECremophor-free, albumin-engineered nanoparticle formulation ofpaclitaxel (American Pharmaceutical Partners, Schaumberg, 111.), andTAXOTERE doxetaxel (Rhone-Poulenc Rorer, Antony, France); chloranbucil;GEMZAR gemcitabine; 6-thioguanine; mercaptopurine; methotrexate;platinum analogs such as cisplatin, oxaliplatin and carboplatin;vinblastine; platinum; etoposide (VP-16); ifosfamide; mitoxantrone;vincristine; NAVELBINE. vinorelbine; novantrone; teniposide; edatrexate;daunomycin; aminopterin; xeloda; ibandronate; irinotecan (Camptosar,CPT-11) (including the treatment regimen of irinotecan with 5-FU andleucovorin); topoisomerase inhibitor RFS 2000; difluoromethylornithine(DMFO); retinoids such as retinoic acid; capecitabine; combretastatin;leucovorin (LV); oxaliplatin, including the oxaliplatin treatmentregimen (FOLFOX); lapatinib (Tykerb); inhibitors of PKC-α, Raf, H-Ras,EGFR (e.g., erlotinib (Tarceva)) and VEGF-A that reduce cellproliferation and pharmaceutically acceptable salts, acids orderivatives of any of the above. In addition, the methods of treatmentcan further include the use of radiation. In addition, the methods oftreatment can further include the use of photodynamic therapy.

In some embodiments, the additional therapeutic agent is anantidiarrheal agent. Antidiarrheal agents suitable for use in thepresent invention include, but are not limited to, DPP-IV inhibitors,natural opioids, such as tincture of opium, paregoric, and codeine,synthetic opioids, such as diphenoxylate, difenoxin and loperamide,bismuth subsalicylate, lanreotide, vapreotide and octreotide, motilnantagonists, COX2 inhibitors like celecoxib, glutamine, thalidomide andtraditional antidiarrheal remedies, such as kaolin, pectin, berberineand muscarinic agents.

In some embodiments, inclusive, without limitation, of autoimmmuneapplications, the additional therapeutic agent is an immunosuppressiveagent. In some embodiments, the immunosuppressive agent is ananti-inflammatory agent such as a steroidal anti-inflammatory agent or anon-steroidal anti-inflammatory agent (NSAID). Steroids, particularlythe adrenal corticosteroids and their synthetic analogues, are wellknown in the art. Examples of corticosteroids useful in the presentinvention include, without limitation, hydroxyltriamcinolone,alpha-methyl dexamethasone, beta-methyl betamethasone, beclomethasonedipropionate, betamethasone benzoate, betamethasone dipropionate,betamethasone valerate, clobetasol valerate, desonide, desoxymethasone,dexamethasone, diflorasone diacetate, diflucortolone valerate,fluadrenolone, fluclorolone acetonide, flumethasone pivalate,fluosinolone acetonide, fluocinonide, flucortine butylester,fluocortolone, fluprednidene (fluprednylidene) acetate, flurandrenolone,halcinonide, hydrocortisone acetate, hydrocortisone butyrate,methylprednisolone, triamcinolone acetonide, cortisone, cortodoxone,flucetonide, fludrocortisone, difluorosone diacetate, fluradrenoloneacetonide, medrysone, amcinafel, amcinafide, betamethasone and thebalance of its esters, chloroprednisone, clocortelone, clescinolone,dichlorisone, difluprednate, flucloronide, flunisolide, fluoromethalone,fluperolone, fluprednisolone, hydrocortisone, meprednisone,paramethasone, prednisolone, prednisone, beclomethasone dipropionate.(NSAIDS) that may be used in the present invention, include but are notlimited to, salicylic acid, acetyl salicylic acid, methyl salicylate,glycol salicylate, salicylmides, benzyl-2,5-diacetoxybenzoic acid,ibuprofen, fulindac, naproxen, ketoprofen, etofenamate, phenylbutazone,and indomethacin. In some embodiments, the immunosupressive agent may becytostatics such as alkylating agents, antimetabolites (e.g.,azathioprine, methotrexate), cytotoxic antibiotics, antibodies (e.g.,basiliximab, daclizumab, and muromonab), anti-immunophilins (e.g.,cyclosporine, tacrolimus, sirolimus), inteferons, opioids, TNF bindingproteins, mycophenolates, and small biological agents (e.g., fingolimod,myriocin). Additional anti-inflammatory agents are described, forexample, in U.S. Pat. No. 4,537,776, the entire contents of which areincorporated by reference herein.

In some embodiments, inclusive of, without limitation, infectiousdisease applications, the present invention pertains to anti-infectivesas additional therapeutic agents. In some embodiments, theanti-infective is an antiviral agent including, but not limited to,Abacavir, Acyclovir, Adefovir, Amprenavir, Atazanavir, Cidofovir,Darunavir, Delavirdine, Didanosine, Docosanol, Efavirenz, Elvitegravir,Emtricitabine, Enfuvirtide, Etravirine, Famciclovir, and Foscarnet. Insome embodiments, the anti-infective is an anti-bacterial agentincluding, but not limited to, cephalosporin antibiotics (cephalexin,cefuroxime, cefadroxil, cefazolin, cephalothin, cefaclor, cefamandole,cefoxitin, cefprozil, and ceftobiprole); fluoroquinolone antibiotics(cipro, Levaquin, floxin, tequin, avelox, and norflox); tetracyclineantibiotics (tetracycline, minocycline, oxytetracycline, anddoxycycline); penicillin antibiotics (amoxicillin, ampicillin,penicillin V, dicloxacillin, carbenicillin, vancomycin, andmethicillin); monobactam antibiotics (aztreonam); and carbapenemantibiotics (ertapenem, doripenem, imipenem/cilastatin, and meropenem).In some embodiments, the anti-infectives include anti-malarial agents(e.g., chloroquine, quinine, mefloquine, primaquine, doxycycline,artemether/lumefantrine, atovaquone/proguanil andsulfadoxine/pyrimethamine), metronidazole, tinidazole, ivermectin,pyrantel pamoate, and albendazole.

In some embodiments, the present invention pertains to various agentsused for treating obesity as additional therapeutic agents. Illustrativeagents used for treating obesity include, but are not limited to,orlistat (e.g. ALL1, XENICAL), loracaserin (e.g. BELVIQ),phentermine-topiramate (e.g. QSYMIA), sibutramme (e.g. REDUCTIL orMERJDIA), rimonabant (ACOMPLLA), exenatide (e.g. BYETTA), pramlintide(e.g. SYMLIN) phentermine, benzphetamine, diethylpropion,phendimetrazme, bupropion, and metformin. Agents that interfere with thebody's ability to absorb specific nutrients in food are among theadditional agents, e.g. orlistat (e.g. ALU, XENICAL), glucomannan, andguar gum. Agents that suppress apetite are also among the additionalagents, e.g. catecholamines and their derivatives (such as phenteimineand other amphetamine-based drugs), various antidepressants and moodstabilizers (e.g. bupropion and topiramate), anorectics (e.g. dexedrine,digoxin). Agents that increase the body's metabolism are also among theadditional agents.

In some embodiments, additional therapeutic agents may be selected fromamong appetite suppressants, neurotransmitter reuptake inhibitors,dopaminergic agonists, serotonergic agonists, modulators of GABAergicsignaling, anticonvulsants, antidepressants, monoamine oxidaseinhibitors, substance P (NK1) receptor antagonists, melanocortinreceptor agonists and antagonists, lipase inhibitors, inhibitors of fatabsorption, regulators of energy intake or metabolism, cannabinoidreceptor modulators, agents for treating addiction, agents for treatingmetabolic syndrome, peroxisome proliferator-activated receptor (PPAR)modulators; dipcptidyl peptidase 4 (DPP-4) antagonists, agents fortreating cardiovascular disease, agents for treating elevatedtriglyceride levels, agents for treating low HDL, agents for treatinghypercholesterolemia, and agents for treating hypertension. Some agentsfor cardiovascular disease include statins (e.g. lovastatin,atorvastatin, fluvastatin, rosuvastatin, simvastatin and pravastatin)and omega-3 agents (e.g. LOVAZA, EPANQVA, VASCEPA, esterified omega-3'sin general, fish oils, krill oils, algal oils). In some embodiments,additional agents may be selected from among amphetamines,benzodiazepines, suifonyl ureas, meglitinides, thiazolidinediones,biguanides, beta-blockers, XCE inhibitors, diuretics, nitrates, calciumchannel blockers, phenlermine, sibutramine, iorcaserin, cetilistat,rimonabant, taranabant, topiramate, gabapentin, valproate, vigabatrin,bupropion, tiagabine, sertraline, fluoxetine, trazodone, zonisamide,methylphenidate, varenicline, naltrexone, diethylpropion,phendimetrazine, rcpaglini.de, nateglinide, glimepiride, metformin,pioglitazone, rosiglilazone, and sitagliptin.

In some embodiments, the present invention pertains to an agent used fortreating diabetes as additional therapeutic agents. Illustrativeanti-diabetic agents include, but are not limited to, sulfonylurea (e.g.DYMELOR (acetohexamide), DIABINESE (chlorpropamide), ORINASE(tolbutamide), and TOLINASE (tolazamide), GLUCOTROL (glipizide),GLUCOTROL XL (extended release), DIABETA (glyburide), MICRONASE(glyburide), GLYNASE PRESTAB (glyburide), and AMARYL (glimepiride)); aBiguanide (e.g. metformin (GLUCOPHAGE, GLUCOPHAGE XR, RIOMET, FORTAMET,and GLUMETZA)); a thiazolidinedione (e.g. ACTOS (pioglitazone) andAVANDIA (rosiglitazone); an alpha-glucosidase inhibitor (e.g., PRECOSE(acarbose) and GLYSET (miglitol); a Meglitinide (e.g., PRANDIN(repaglinide) and STARLIX (nateglinide)); a Dipeptidyl peptidase IV(DPP-IV) inhibitor (e.g., JANUVIA (sitagliptin), NESINA (alogliptin),ONGLYZA (saxagliptin), and TRADJENTA (linagliptin)); Sodium-glucoseco-transporter 2 (SGLT2) inhibitor (e.g. INVOKANA (canaglifozin)); and acombination pill (e.g. GLUCOVANCE, which combines glyburide (asulfonylurea) and metformin, METAGLIP, which combines glipizide (asulfonylurea) and metformin, and AVANDAMET, which uses both metforminand rosiglitazone (AVANDIA) in one pill, KAZANO (alogliptin andmetformin), OSENI (alogliptin plus pioglitazone), METFORMIN oral, ACTOSoral, BYETTA subcutaneous, JANUVIA oral, WELCHOL oral, JANUMET oral,glipizide oral, glimepiride oral, GLUCOPHAGE oral, LANTUS subcutaneous,glyburide oral, ONGLYZA oral, AMARYI oral, LANTUS SOLOSTAR subcutaneous,BYDUREON subcutaneous, LEVEMIR FLEXPEN subcutaneous, ACTOPLUS MET oral,GLUMETZA oral, TRADJENTA oral, bromocriptine oral, KOMBIGLYZE XR oral,INVOKANA oral, PRANDIN oral, LEVEMIR subcutaneous, PARLODEL oral,pioglitazone oral, NOVOLOG subcutaneous, NOVOLOG FLEXPEN subcutaneous,VICTOZA 2-PAK subcutaneous, HUMALOG subcutaneous, STARLIX oral, FORTAMEToral, GLUCOVANCE oral, GLUCOPHAGE XR oral, NOVOLOG Mix 70-30 FLEXPENsubcutaneous, GLYBURIDE-METFORMIN oral, acarbose oral, SYMLINPEN 60subcutaneous, GLUCOTROI XL oral, NOVOLIN R inj, GLUCOTROL oral, DUETACToral, sitagliptin oral, SYMLINPEN 120 subcutaneous, HUMALOG KWIKPENsubcutaneous, JANUMET XR oral, GLIPIZIDE-METFORMIN oral, CYCLOSET oral,HUMALOG MIX 75-25 subcutaneous, nateglinide oral, HUMALOG Mix 75-25KWIKPEN subcutaneous, HUMULIN 70/30 subcutaneous, PRECOSE oral, APIDRAsubcutaneous, Humulin R inj, Jentadueto oral, Victoza 3-Paksubcutaneous, Novolin 70/30 subcutaneous, NOVOLIN N subcutaneous,insulin detemir subcutaneous, glyburide micronized oral, GLYNASE oral,HUMULIN N subcutaneous, insulin glargine subcutaneous, RIOMET oral,pioglitazone-metformin oral, APIDRA SOLOSTAR subcutaneous, insulinlispro subcutaneous, GLYSET oral, HUMULIN 70/30 Pen subcutaneous,colesevelam oral, sitagliptin-metformin oral, DIABETA oral, insulinregular human inj, HUMULIN N Pen subcutaneous, exenatide subcutaneous,HUMALOG Mix 50-50 KWIKPEN subcutaneous, liraglutide subcutaneous, KAZANOoral, repaglinide oral, chlorpropamide oral, insulin aspartsubcutaneous, NOVOLOG Mix 70-30 subcutaneous, HUMALOG Mix 50-50subcutaneous, saxagliptin oral, ACTOPLUS Met XR oral, miglitol oral, NPHinsulin human recomb subcutaneous, insulin NPH and regular humansubcutaneous, tolazamide oral, mifepristone oral, insulin aspartprotam-insulin aspart subcutaneous, repaglinide-metformin oral,saxagliptin-metformin oral, linagliptin-metformin oral, NESINA oral,OSENI oral, tolbutamide oral, insulin lispro protamine and lisprosubcutaneous, pramlintide subcutaneous, insulin glulisine subcutaneous,pioglitazone-glimepiride oral, PRANDIMET oral, NOVOLOG PenFillsubcutaneous, linagliptin oral, exenatide microspheres subcutaneous,KORLYM oral, alogliptin oral, alogliptin-pioglitazone oral,alogliptin-metformin oral, canagliflozin oral, Lispro (HUMALOG); Aspart(NOVOLOG); Glulisine (APIDRA); Regular (NOVOLIN R or HUMULIN R); NPH(NOVOLIN N or HUMULIN N); Glargine (LANTUS); Detemir (LEVEMIR); HUMULINor NOVOLIN 70/30; and NOVOLOG Mix 70/30 HUMALOG Mix 75/25 or 50/50.

In some embodiments, the present invention relates to combinationtherapy with a blood transfusion. For instance, the present compositionsmay supplement a blood transfusion. In some embodiments, the presentinvention relates to combination therapy with iron supplements.

In some embodiments, the present invention relates to the use of one ormore EPO-based agents as additional therapeutic agents. For example, thepresent compositions can be used as an adjuvant to other EPO-basedagents. In some embodiments, the present compositions are used as amaintenance therapy to other EPO-based agents. Other EPO-based agentsinclude the following: epoetin alfa, including without limitation,DARBEPOETIN (ARANESP), EPOCEPT (LUPIN PHARMA), NANOKINE (NANOGENPHARMACEUTICAL), EPOFIT (INTAS PHARMA), EPOGEN (AMGEN), EPOGIN, EPREX,(JANSSEN-CILAG), BINOCRIT (SANDOZ), PROCRIT; epoetin beta, includingwithout limitation, NEORECORMON (HOFFMANN-LA ROCHE), RECORMON, Methoxypolyethylene glycol-epoetin beta (MIRCERA, ROCHE); epoetin delta,including without limitation, DYNEPO (erythropoiesis stimulatingprotein, SHIRE PLC); epoetin omega, including without limitation,EPOMAX; epoetin zeta, including without limitation, SILAPO (STADA) andRETACRIT (HOSPIRA) and other EPOs, including without limitation, EPOCEPT(LUPIN PHARMACEUTICALS), EPOTRUST (PANACEA BIOTEC LTD), ERYPRO SAFE(BIOCON LTD.), REPOITIN (SERUM INSTITUTE OF INDIA LIMITED), VINTOR(EMCURE PHARMACEUTICALS), EPOFIT (INTAS PHARMA), ERYKINE (INTASBIOPHARMACEUTICA), WEPDX (WOCKHARDT BIOTECH), ESPOGEN (LG LIFESCIENCES), RELIPOIETIN (RELIANCE LIFE SCIENCES), SHANPOIETIN (SHANTHABIOTECHNICS LTD), ZYROP (CADILA HEALTHCARE LTD.), EPIAO (RHUEPO)(SHENYANG SUNSHINE PHARMACEUTICAL CO. LTD), CINNAPOIETIN (CINNAGEN).

In some embodiments, the chimeric protein described herein, includederivatives that are modified, i.e., by the covalent attachment of anytype of molecule to the composition such that covalent attachment doesnot prevent the activity of the composition. For example, but not by wayof limitation, derivatives include composition that have been modifiedby, inter alia, glycosylation, lipidation, acetylation, pegylation,phosphorylation, amidation, derivatization by known protecting/blockinggroups, proteolytic cleavage, linkage to a cellular ligand or otherprotein, etc. Any of numerous chemical modifications can be carried outby known techniques, including, but not limited to specific chemicalcleavage, acetylation, formylation, metabolic synthesis of tunicamycin,etc.

In still other embodiments, the chimeric protein described hereinfurther comprise a cytotoxic agent, comprising, in illustrativeembodiments, a toxin, a chemotherapeutic agent, a radioisotope, and anagent that causes apoptosis or cell death. Such agents may be conjugatedto a composition described herein.

The chimeric protein described herein may thus be modifiedpost-translationally to add effector moieties such as chemical linkers,detectable moieties such as for example fluorescent dyes, enzymes,substrates, bioluminescent materials, radioactive materials, andchemiluminescent moieties, or functional moieties such as for examplestreptavidin, avidin, biotin, a cytotoxin, a cytotoxic agent, andradioactive materials. In an embodiment, the effector moiety is a Histag.

Illustrative cytotoxic agents include, but are not limited to,methotrexate, aminopterin, 6-mercaptopurine, 6-thioguanine, cytarabine,5-fluorouracil decarbazine; alkylating agents such as mechlorethamine,thioepa chlorambucil, melphalan, carmustine (BSNU), mitomycin C,lomustine (CCNU), 1-methylnitrosourea, cyclothosphamide,mechlorethamine, busulfan, dibromomannitol, streptozotocin, mitomycin C,cis-dichlorodiamine platinum (II) (DDP) cisplatin and carboplatin(paraplatin); anthracyclines include daunorubicin (formerly daunomycin),doxorubicin (adriamycin), detorubicin, carminomycin, idarubicin,epirubicin, mitoxantrone and bisantrene; antibiotics includedactinomycin (actinomycin D), bleomycin, calicheamicin, mithramycin, andanthramycin (AMC); and antimytotic agents such as the vinca alkaloids,vincristine and vinblastine. Other cytotoxic agents include paclitaxel(taxol), ricin, pseudomonas exotoxin, gemcitabine, cytochalasin B,gramicidin D, ethidium bromide, emetine, etoposide, tenoposide,colchicin, dihydroxy anthracin dione, 1-dehydrotestosterone,glucocorticoids, procaine, tetracaine, lidocaine, propranolol,puromycin, procarbazine, hydroxyurea, asparaginase, corticosteroids,mytotane (O,P′-(DDD)), interferons, and mixtures of these cytotoxicagents.

Further cytotoxic agents include, but are not limited to,chemotherapeutic agents such as carboplatin, cisplatin, paclitaxel,gemcitabine, calicheamicin, doxorubicin, 5-fluorouracil, mitomycin C,actinomycin D, cyclophosphamide, vincristine, bleomycin, VEGFantagonists, EGFR antagonists, platins, taxols, irinotecan,5-fluorouracil, gemcytabine, leucovorine, steroids, cyclophosphamide,melphalan, vinca alkaloids (e.g., vinblastine, vincristine, vindesineand vinorelbine), mustines, tyrosine kinase inhibitors, radiotherapy,sex hormone antagonists, selective androgen receptor modulators,selective estrogen receptor modulators, PDGF antagonists, TNFantagonists, IL-1 antagonists, interleukins (e.g. IL-12 or IL-2), IL-12Rantagonists, Toxin conjugated monoclonal antibodies, tumor antigenspecific monoclonal antibodies, Erbitux, Avastin, Pertuzumab, anti-CD20antibodies, Rituxan, ocrelizumab, ofatumumab, DXL625, HERCEPTIN®, or anycombination thereof. Toxic enzymes from plants and bacteria such asricin, diphtheria toxin and Pseudomonas toxin may be conjugated to thetherapeutic agents (e.g. antibodies) to generatecell-type-specific-killing reagents (Youle, et al., Proc. Nat'l Acad.Sci. USA 77:5483 (1980); Gilliland, et al., Proc. Nat'l Acad. Sci. USA77:4539 (1980); Krolick, et al., Proc. Nat'l Acad. Sci. USA 77:5419(1980)).

Other cytotoxic agents include cytotoxic ribonucleases as described byGoldenberg in U.S. Pat. No. 6,653,104. Embodiments of the invention alsorelate to radioimmunoconjugates where a radionuclide that emits alpha orbeta particles is stably coupled to the chimeric protein, with orwithout the use of a complex-forming agent. Such radionuclides includebeta-emitters such as Phosphorus-32, Scandium-47, Copper-67, Gallium-67,Yttrium-88, Yttrium-90, Iodine-125, Iodine-131, Samarium-153,Lutetium-177, Rhenium-186 or Rhenium-188, and alpha-emitters such asAstatine-211, Lead-212, Bismuth-212, Bismuth-213 or Actinium-225.

Illustrative detectable moieties further include, but are not limitedto, horseradish peroxidase, acetylcholinesterase, alkaline phosphatase,beta-galactosidase and luciferase. Further illustrative fluorescentmaterials include, but are not limited to, rhodamine, fluorescein,fluorescein isothiocyanate, umbelliferone, dichlorotriazinylamine,phycoerythrin and dansyl chloride. Further illustrative chemiluminescentmoieties include, but are not limited to, luminol. Further illustrativebioluminescent materials include, but are not limited to, luciferin andaequorin. Further illustrative radioactive materials include, but arenot limited to, Iodine-125, Carbon-14, Sulfur-35, Tritium andPhosphorus-32.

Methods of Treatment

Methods and compositions described herein have broad application totreating various diseases and disorders, including, but not limited tocancer, infections, immune disorders, autoimmune diseases,cardiovascular diseases, wound healing, ischemia-related diseases,neurodegenerative diseases, metabolic diseases, anemia, and many otherdiseases and disorders.

In some embodiments, the present invention relates to the treatment of,or a patient having one or more of cancer, heart failure, autoimmunedisease, sickle cell disease, thalassemia, blood loss, transfusionreaction, diabetes, vitamin B12 deficiency, collagen vascular disease,Shwachman syndrome, thrombocytopenic purpura, Celiac disease, endocrinedeficiency state such as hypothyroidism or Addison's disease, autoimmunedisease such as Crohn's Disease, systemic lupus erythematosis,rheumatoid arthritis or juvenile rheumatoid arthritis, ulcerativecolitis immune disorders such as eosinophilic fasciitis,hypoimmunoglobulinemia, or thymoma/thymic carcinoma, graft versus hostdisease, preleukemia, Nonhematologic syndrome (e.g. Down's, Dubowwitz,Seckel), Felty syndrome, hemolytic uremic syndrome, myelodysplasicsyndrome, nocturnal paroxysmal hemoglobinuria, osteomyelofibrosis,pancytopenia, pure red-cell aplasia, Schoenlein-Henoch purpura, malaria,protein starvation, menorrhagia, systemic sclerosis, liver cirrhosis,hypometabolic states, congestive heart failure, chronic infections suchas HIV/AIDS, tuberculosis, oseomyelitis, hepatitis B, hepatitis C,Epstein-barr virus or parvovirus, T cell leukemia virus, bacterialovergrowth syndrome, fungal or parasitic infections, and/or red cellmembrane disorders such as hereditary spherocytosis, hereditaryelliptocytosis, hereditary pyrpoikilocytosis, hereditary stomatocytosis,red cell enzyme defects, hypersplenism, immune hemolysis or paroxysmalnocturnal hemoglobinuria.

In some embodiments, the present invention relates to the treatment of,or a patient having cancer. As used herein, cancer refers to anyuncontrolled growth of cells that may interfere with the normalfunctioning of the bodily organs and systems, and includes both primaryand metastatic tumors. Primary tumors or cancers that migrate from theiroriginal location and seed vital organs can eventually lead to the deathof the subject through the functional deterioration of the affectedorgans. A metastasis is a cancer cell or group of cancer cells, distinctfrom the primary tumor location, resulting from the dissemination ofcancer cells from the primary tumor to other parts of the body.Metastases may eventually result in death of a subject. For example,cancers can include benign and malignant cancers, polyps, hyperplasia,as well as dormant tumors or micrometastases.

Illustrative cancers that may be treated include, but are not limitedto, basal cell carcinoma, biliary tract cancer; bladder cancer; bonecancer; brain and central nervous system cancer; breast cancer; cancerof the peritoneum; cervical cancer; choriocarcinoma; colon and rectumcancer; connective tissue cancer; cancer of the digestive system;endometrial cancer; esophageal cancer; eye cancer; cancer of the headand neck; gastric cancer (including gastrointestinal cancer);glioblastoma; hepatic carcinoma; hepatoma; intra-epithelial neoplasm;kidney or renal cancer; larynx cancer; leukemia; liver cancer; lungcancer (e.g., small-cell lung cancer, non-small cell lung cancer,adenocarcinoma of the lung, and squamous carcinoma of the lung);melanoma; myeloma; neuroblastoma; oral cavity cancer (lip, tongue,mouth, and pharynx); ovarian cancer; pancreatic cancer; prostate cancer;retinoblastoma; rhabdomyosarcoma; rectal cancer; cancer of therespiratory system; salivary gland carcinoma; sarcoma; skin cancer;squamous cell cancer; stomach cancer; testicular cancer; thyroid cancer;uterine or endometrial cancer; cancer of the urinary system; vulvalcancer; lymphoma including Hodgkin's and non-Hodgkin's lymphoma, as wellas B-cell lymphoma (including low grade/follicular non-Hodgkin'slymphoma (NHL); small lymphocytic (SL) NHL; intermediategrade/follicular NHL; intermediate grade diffuse NHL; high gradeimmunoblastic NHL; high grade lymphoblastic NHL; high grade smallnon-cleaved cell NHL; bulky disease NHL; mantle cell lymphoma;AIDS-related lymphoma; and Waldenstrom's Macroglobulinemia; chroniclymphocytic leukemia (CLL); acute lymphoblastic leukemia (ALL); Hairycell leukemia; chronic myeloblastic leukemia; as well as othercarcinomas and sarcomas; and post-transplant lymphoproliferativedisorder (PTLD), as well as abnormal vascular proliferation associatedwith phakomatoses, edema (e.g. that associated with brain tumors), andMeigs' syndrome.

In some embodiments, the present invention relates to the treatment of,or a patient having a microbial infection and/or chronic infection.Illustrative infections include, but are not limited to, HIV/AIDS,tuberculosis, osteomyelitis, hepatitis B, hepatitis C, Epstein-barrvirus or parvovirus, T cell leukemia virus, bacterial overgrowthsyndrome, fungal or parasitic infections.

In various embodiments, the present compositions are used to treat orprevent one or more inflammatory diseases or conditions, such asinflammation, acute inflammation, chronic inflammation, respiratorydisease, atherosclerosis, restenosis, asthma, allergic rhinitis, atopicdermatitis, septic shock, rheumatoid arthritis, inflammatory boweldisease, inflammatory pelvic disease, pain, ocular inflammatory disease,celiac disease, Leigh Syndrome, Glycerol Kinase Deficiency, Familialeosinophilia (FE), autosomal recessive spastic ataxia, laryngealinflammatory disease; Tuberculosis, Chronic cholecystitis,Bronchiectasis, Silicosis and other pneumoconioses.

In various embodiments, the present compositions are used to treat orprevent one or more autoimmune diseases or conditions, such as multiplesclerosis, diabetes mellitus, lupus, celiac disease, Crohn's disease,ulcerative colitis, Guillain-Barre syndrome, scleroderms, Goodpasture'ssyndrome, Wegener's granulomatosis, autoimmune epilepsy, Rasmussen'sencephalitis, Primary biliary sclerosis, Sclerosing cholangitis,Autoimmune hepatitis, Addison's disease, Hashimoto's thyroiditis,Fibromyalgia, Menier's syndrome; transplantation rejection (e.g.,prevention of allograft rejection) pernicious anemia, rheumatoidarthritis, systemic lupus erythematosus, dermatomyositis, Sjogren'ssyndrome, lupus erythematosus, multiple sclerosis, myasthenia gravis,Reiter's syndrome, Grave's disease, and other autoimmune diseases.

In various embodiments, the present compositions are used to treat,control or prevent cardiovascular disease, such as a disease orcondition affecting the heart and vasculature, including but not limitedto, coronary heart disease (CHD), cerebrovascular disease (CVD), aorticstenosis, peripheral vascular disease, atherosclerosis,arteriosclerosis, myocardial infarction (heart attack), cerebrovasculardiseases (stroke), transient ischaemic attacks (TIA), angina (stable andunstable), atrial fibrillation, arrhythmia, valvular disease, and/orcongestive heart failure.

In various embodiments, the present compositions are used to treat orprevent one or more metabolic-related disorders. In various embodiments,the present invention is useful for the treatment, controlling orprevention of diabetes, including Type 1 and Type 2 diabetes anddiabetes associated with obesity.

The compositions and methods of the present invention are useful for thetreatment or prevention of diabetes-related disorders, including withoutlimitation diabetic nephropathy, hyperglycemia, impaired glucosetolerance, insulin resistance, obesity, lipid disorders, dyslipidemia,hyperlipidemia, hypertriglyceridemia, hypercholesterolemia, low HDLlevels, high LDL levels, atherosclerosis and its sequelae, vascularrestenosis, irritable bowel syndrome, inflammatory bowel disease,including Crohn's disease and ulcerative colitis, other inflammatoryconditions, pancreatitis, abdominal obesity, neurodegenerative disease,retinopathy, neoplastic conditions, adipose cell tumors, adipose cellcarcinomas, such as liposarcoma, prostate cancer and other cancers,including gastric, breast, bladder and colon cancers, angiogenesis,Alzheimer's disease, psoriasis, high blood pressure, Metabolic Syndrome(e.g. a person has three or more of the following disorders: abdominalobesity, hypertriglyceridemia, low HDL cholesterol, high blood pressure,and high fasting plasma glucose), ovarian hyperandrogenism (polycysticovary syndrome), and other disorders where insulin resistance is acomponent, such as sleep apnea.

The compositions and methods of the present invention are useful for thetreatment, control, or prevention of obesity, including genetic orenvironmental, and obesity-related disorders. The obesity-relateddisorders herein are associated with, caused by, or result from obesity.Examples of obesity-related disorders include obesity, diabetes,overeating, binge eating, and bulimia, hypertension, elevated plasmainsulin concentrations and insulin resistance, dyslipidemia,hyperlipidemia, endometrial, breast, prostate, kidney and colon cancer,osteoarthritis, obstructive sleep apnea, gallstones, heart disease,abnormal heart rhythms and arrythmias, myocardial infarction, congestiveheart failure, coronary heart disease, sudden death, stroke, polycysticovary disease, craniopharyngioma, Prader-Willi Syndrome, Frohlich'ssyndrome, GH-deficient subjects, normal variant short stature, Turner'ssyndrome, and other pathological conditions showing reduced metabolicactivity or a decrease in resting energy expenditure as a percentage oftotal fat-free mass, e.g, children with acute lymphoblastic leukemia.Further examples of obesity-related disorders are Metabolic Syndrome,insulin resistance syndrome, reproductive hormone abnormalities, sexualand reproductive dysfunction, such as impaired fertility, infertility,hypogonadism in males and hirsutism in females, fetal defects associatedwith maternal obesity, gastrointestinal motility disorders, such asobesity-related gastro-esophageal reflux, respiratory disorders, such asobesity-hypoventilation syndrome (Pickwickian syndrome), breathlessness,cardiovascular disorders, inflammation, such as systemic inflammation ofthe vasculature, arteriosclerosis, hypercholesterolemia, lower backpain, gallbladder disease, hyperuricemia, gout, and kidney cancer, andincreased anesthetic risk. The compositions and methods of the presentinvention are also useful to treat Alzheimer's disease.

In various embodiments, the present compositions are used to treat orprevent one or more respiratory diseases, such as asthma, chronicobstructive pulmonary disease (COPD), bronchiectasis, allergic rhinitis,sinusitis, pulmonary vasoconstriction, inflammation, allergies, impededrespiration, respiratory distress syndrome, cystic fibrosis, pulmonaryhypertension, pulmonary vasoconstriction, emphysema, Hantaviruspulmonary syndrome (HPS), Loeffler's syndrome, Goodpasture's syndrome,Pleurisy, pneumonitis, pulmonary edema, pulmonary fibrosis, Sarcoidosis,complications associated with respiratory syncitial virus infection, andother respiratory diseases.

In some embodiments, the present invention is used to treat or preventone or more neurodegenerative disease. Illustrative neurodegenerativedisease include, but are not limited to, multiple sclerosis (includingwithout limitation, benign multiple sclerosis; relapsing-remittingmultiple sclerosis (RRMS); secondary progressive multiple sclerosis(SPMS); progressive relapsing multiple sclerosis (PRMS); and primaryprogressive multiple sclerosis (PPMS)), Alzheimer's. disease (including,without limitation, Early-onset Alzheimer's, Late-onset Alzheimer's, andFamilial Alzheimer's disease (FAD), Parkinson's disease and parkinsonism(including, without limitation, Idiopathic Parkinson's disease, Vascularparkinsonism, Drug-induced parkinsonism, Dementia with Lewy bodies,Inherited Parkinson's, Juvenile Parkinson's), Huntington's disease,Amyotrophic lateral sclerosis (ALS, including, without limitation,Sporadic ALS, Familial ALS, Western Pacific ALS, Juvenile ALS, HiramayaDisease).

In various embodiments, the present chimeric proteins find use intreating wounds, e.g., a non-healing wound, an ulcer, a burn, orfrostbite, a chronic or acute wound, open or closed wound, internal orexternal wound (illustrative external wounds are penetrating andnon-penetrating wound. In various embodiments, the present chimericproteins find use in treating ischemia, by way of non-limiting example,ischemia associated with acute coronary syndrome, acute lung injury(ALI), acute myocardial infarction (AMI), acute respiratory distresssyndrome (ARDS), arterial occlusive disease, arteriosclerosis, articularcartilage defect, aseptic systemic inflammation, atheroscleroticcardiovascular disease, autoimmune disease, bone fracture, bonefracture, brain edema, brain hypoperfusion, Buerger's disease, burns,cancer, cardiovascular disease, cartilage damage, cerebral infarct,cerebral ischemia, cerebral stroke, cerebrovascular disease,chemotherapy-induced neuropathy, chronic infection, chronic mesentericischemia, claudication, congestive heart failure, connective tissuedamage, contusion, coronary artery disease (CAD), critical limb ischemia(CLI), Crohn's disease, deep vein thrombosis, deep wound, delayed ulcerhealing, delayed wound-healing, diabetes (type I and type II), diabeticneuropathy, diabetes induced ischemia, disseminated intravascularcoagulation (DIC), embolic brain ischemia, frostbite, graft-versus-hostdisease, hereditary hemorrhagic telengiectasiaischemic vascular disease,hyperoxic injury, hypoxia, inflammation, inflammatory bowel disease,inflammatory disease, injured tendons, intermittent claudication,intestinal ischemia, ischemia, ischemic brain disease, ischemic heartdisease, ischemic peripheral vascular disease, ischemic placenta,ischemic renal disease, ischemic vascular disease, ischemic-reperfusioninjury, laceration, left main coronary artery disease, limb ischemia,lower extremity ischemia, myocardial infarction, myocardial ischemia,organ ischemia, osteoarthritis, osteoporosis, osteosarcoma, Parkinson'sdisease, peripheral arterial disease (PAD), peripheral artery disease,peripheral ischemia, peripheral neuropathy, peripheral vascular disease,pre-cancer, pulmonary edema, pulmonary embolism, remodeling disorder,renal ischemia, retinal ischemia, retinopathy, sepsis, skin ulcers,solid organ transplantation, spinal cord injury, stroke,subchondral-bone cyst, thrombosis, thrombotic brain ischemia, tissueischemia, transient ischemic attack (TIA), traumatic brain injury,ulcerative colitis, vascular disease of the kidney, vascularinflammatory conditions, von Hippel-Lindau syndrome, or wounds totissues or organs.

In various embodiments pertaining to the use of chimeric proteins havingEPO as the modified soluble agent, the present invention relates to thetreatment of one or more of anemia, including anemia resulting fromchronic kidney disease (e.g. from dialysis) and/or an anti-cancer agent(e.g. chemotherapy and/or HIV treatment (e.g. Zidovudine (INN) orazidothymidine (AZT)), inflammatory bowel disease (e.g. Crohn's diseaseand ulcer colitis), anemia linked to inflammatory conditions (e.g.arthritis, lupus, IBD), anemia linked to diabetes, schizophrenia,cerebral malaria, as aplastic anemia, and myelodysplasia from thetreatment of cancer (e.g. chemotherapy and/or radiation), and variousmyelodysplastic syndrome diseases (e.g. sickle cell anemia, hemoglobinSC disease, hemoglobin C disease, alpha- and beta-thalassemias, neonatalanemia after premature birth, and comparable conditions).

In some embodiments, the present invention relates to the treatment of,or a patient having anemia, i.e. a condition in which the number of redblood cells and/or the amount of hemoglobin found in the red blood cellsis below normal. In various embodiments, the anemia may be acute orchronic. For example, the present anemias include but are not limited toiron deficiency anemia, renal anemia, anemia of chronicdiseases/inflammation, pernicious anemia such as macrocytic achylicanemia, juvenile pernicious anemia and congenital pernicious anemia,cancer-related anemia, anti-cancer-related anemia (e.g.chemotherapy-related anemia, radiotherapy-related anemia), pure red cellaplasia, refractory anemia with excess of blasts, aplastic anemia,X-lined siderobalstic anemia, hemolytic anemia, sickle cell anemia,anemia caused by impaired production of ESA, myelodysplasia syndromes,hypochromic anemia, microcytic anemia, sideroblastic anemia, autoimmunehemolytic anemia, Cooley's anemia, Mediterranean anemia, DiamondBlackfan anemia, Fanconi's anemia and drug-induced immune hemolyticanemia. Anemia may cause serious symptoms, including hypoxia, chronicfatigue, lack of concentration, pale skin, low blood pressure, dizzinessand heart failure.

In some embodiments, the present invention relates to the treatment ofanemia resulting from chronic renal failure. In some embodiments, thepresent invention relates to the treatment of anemia resulting from theuse of one or more renal replacement therapies, inclusive of dialysis,hemodialysis, peritoneal dialysis, hemofiltration, hemodiafiltration,and renal transplantation.

In some embodiments, the present invention relates to the treatment ofanemia in patients with chronic kidney disease who are not on dialysis.For instance, the present invention relates to patients in stage 1 CKD,or stage 2 CKD, or stage 3 CKD, or stage 4 CKD, or stage 5 CKD. In someembodiments, the present patient is stage 4 CKD or stage 5 CKD. In someembodiments, the present patient has undergone a kidney transplant. Insome embodiments, the present invention relates to the treatment ofanemia is a patient having an acute kidney injury (AKI).

In some embodiments, the anemia is induced by chemotherapy. Forinstance, the chemotherapy may be any myelosuppressive chemotherapy. Insome embodiment, the chemotherapy is one or more of Revlimid, Thalomid,dexamethasone, Adriamycin and Doxil. In some embodiments, thechemotherapy is one or more platinum-based drugs including cisplatin(e.g. PLATINOL) and carboplatin (e.g. PARAPLATIN). In some embodiments,the chemotherapy is any one of the chemotherapeutic agents describedherein. In some embodiments, the chemotherapy is any agent described inGroopman et al. J Natl Cancer Inst (1999) 91 (19): 1616-1634, thecontents of which are hereby incorporated by reference in theirentireties. In some embodiments, the present compositions and methodsare used in the treatment of chemotherapy-related anemia in later stagecancer patients (e.g. a stage IV, or stage III, or stage II cancer). Insome embodiments, the present compositions and methods are used in thetreatment of chemotherapy-related anemia in cancer patients receivingdose-dense chemotherapy or other aggressive chemotherapy regimens.

In some embodiments, the present invention relates to the treatment ofanemia in a patient having one or more blood-based cancers, such asleukemia, lymphoma, and multiple myeloma. Such cancers may affect thebone marrow directly. Further, the present invention relates tometastatic cancer that has spread to the bone or bone marrow. In someembodiments, the present invention relates to the treatment of anemia ina patient undergoing radiation therapy. Such radiation therapy maydamage the bone marrow, lowering its ability to make red blood cells. Infurther embodiments, the present invention relates to the treatment ofanemia in a patient having a reduction or deficiency of one or more ofiron, vitamin B12, and folic acid. In further embodiments, the presentinvention relates to the treatment of anemia in a patient havingexcessive bleeding including without limitation, after surgery or from atumor that is causing internal bleeding. In further embodiments, thepresent invention relates to the treatment of anemia in a patient havinganemia of chronic disease.

In various embodiments, the present compositions and methods are used toreduce or eliminate fatigue, dizziness, and shortness of breath in apatient.

In various embodiments, the present compositions and methods are used totreat a patient presenting with hyporesponse or resistance toerythropoiesis stimulating agent therapy. In some embodiments,hyporesponsiveness to erythropoietin or ESA-resistant anemia refers tothe presence of at least one of the following conditions: i) asignificant decrease in hemoglobin levels at a constant dose of ESAtreatment, ii) a significant increase in the ESA dose requirement toachieve or maintain a certain hemoglobin level, iii) a failure to raisethe hemoglobin level to the target range despite the ESA dose equivalentto erythropoietin greater than 150 IU/kg/week or 0.75 mg/kg/week ofdarbepoeitn-alpha or continued need for such high dose of ESA tomaintain the target hemoglobin level. For example, approximately 5-10%of patients with CDK demonstrate hyporesponsiveness to ESA, defined as acontinued need for greater than 300 IU/kg per week erythropoietin or 1.5ng/kg per week darbepoetin administered by the subcutaneous route.

In various embodiments, the present compositions and methods mitigatethe need for dose-escalation of the erythropoiesis stimulating agenttherapy and therefore, optionally, avoid side effects (e.g. flu-likesymptoms such as joint pains, weakness, dizziness and tiredness, skinirritation, increased risk of adverse cardiovascular complications).

In various embodiments, the present compositions and methods are used tomaintain a hemoglobin level of about 12.5 to 13 g/dL. In variousembodiments, the present compositions and methods are used in patientshaving hemoglobin levels of below about 12 g/dL, or about 11 g/dL, orabout 10 g/dL, or about 9 g/dL, or about 8 g/dL, or about 7 g/dL, orabout 6 g/dL, or about 5 g/dL. In various embodiments, the presentcompositions and methods are used in patients having iron blood testscores that indicate blood pathology, e.g. a ferritin score of belowabout 200 ng/L and/or a transferrin saturation score below about 30%.

In various embodiments, the present compositions and methods are used toincrease or maintain hemoglobin levels at a target level ranging from 9to 10 g/dL, at a target level ranging from 9 g/dL to 11 g/dL, at atarget level ranging from 9 g/dL to 12 g/dL, at a target level rangingfrom 9 g/dL to 14 g/dL, at a target level ranging from 10 g/dL to 14g/dL, or at a target level ranging from 12 g/dL to 14 g/dL.

In various embodiments, the present compositions and methods are used tobring a patient's hemoglobin levels to normal. In various embodiments,normal hemoglobin ranges for humans are about 14-18 g/dl for men and12-16 for women g/dl with the average hemoglobin value for men at about16 g/dL and for women at about 14 g/dL.

In some embodiments, the present invention relates to the treatment ofanemia of one or more of the following toxicity grading criteria (e.g.NCI Common Toxicity Criteria): grade 1 (mild), 10.0 g hemoglobin/dL towithin normal limits; grade 2 (moderate), 8.0-10.0 g of hemoglobin/dL;grade 3 (serious or severe), 6.5-7.9 g of hemoglobin/dL; and grade 4(life threatening), less than 6.5 g of hemoglobin/dL. In variousembodiments, the present invention brings an increase in toxicitygrading criteria by about 1 point, or about 2 points, or about 3 points,or about 4 points. In various embodiments, the present invention resultsin a patient having a level of 0 or 1. In various embodiments, thepresent compositions and methods improve anemia as assessed by one ormore scales described in Groopman et al. J Natl Cancer Inst (1999) 91(19): 1616-1634, the entire contents of which are hereby incorporated byreference in their entireties.

In various embodiments, the chimeric EPO protein of the inventionstimulates erythropoiesis in patient in whom the endogenous productionof erythropoietin is impaired. As described previously, the chimeric EPOprotein comprises one or more mutations that allow for the chimeric EPOprotein to have one or more of attenuated activity, reduced endogenousactivity, reduced binding affinity, and decreased specific bioactivityrelative to unmutated, i.e. wild type EPO or other EPO-based agentsdescribed herein. Consequentially, in various embodiments,administration of the chimeric EPO protein of the invention results inreduced systemic toxicity, reduced side effects, and reduced off-targeteffects relative to unmutated, i.e., wild type EPO protein or otherEPO-based agents.

In an embodiment, the chimeric EPO protein has a lower side effectprofile as compared to a wild type EPO or other EPO-based agents. Forexamples, administration of the EPO protein results in reducedincidences of flu-like symptoms in patients such as joint pains,weakness, dizziness and tiredness, skin irritation, increased risk ofadverse cardiovascular complications. In an embodiment, the chimeric EPOprotein of the invention reduces a risk of blood clot formation (e.g.thrombosis, venous thromboembolism). In various embodiments, methods ofthe invention provides a reduced likelihood of blood clot formation(e.g. thrombosis, venous thromboembolism) and/or tumor progression orrecurrence (e.g. of breast, non-small cell lung, head and neck,lymphoid, and cervical cancers) relative to wild type EPO and/or anEPO-based agent. In an embodiment, use of the chimeric EPO protein ofthe invention reduces a risk of blood clot formation (e.g. thrombosis,venous thromboembolism). Accordingly, in various embodiments, thechimeric EPO protein of the invention is particularly suitable for useand is administered to a patient having one or more of a history ofblood clots, recent surgery, prolonged periods of bed rest or limitedactivity, and treatment with a chemotherapeutic agent (e.g. Revlimid,Thalomid, dexamethasone, Adriamycin and Doxil).

Because of the length of time often required for erythropoiesis (severaldays for erythroid progenitors to mature and be released into thecirculation), a clinically significant increase in hemoglobin is oftennot observed in less than two weeks and may require up to ten weeks insome patients following the use of wild type EPO or other EPO-basedagents. The present methods and compositions provide, in someembodiments, a more rapid therapeutic effect. In some embodiments, thepresent methods and compositions provide a more rapid therapeuticeffect, for instance, when compared to wild type EPO or other EPO-basedagents. In some embodiments, the present methods and compositionsprovide a sustained therapeutic effect. In some embodiments, the presentmethods and compositions provide a more sustained therapeutic effect,for instance, when compared to wild type EPO or other EPO-based agents.

For example, in some embodiments, the present methods and compositionsprovide a clinically significant increase in hematocrit in less thanabout 6 weeks, or less than about 5 weeks, or less than about 4 weeks,or less than about 3 weeks, or less than about 2 weeks, or less thanabout 1 week. In some embodiments, the present methods and compositionsprovide a clinically significant increase in hematocrit in about 2weeks, or about 10 days, or about 1 week, or about 3 days, or about 1day. In various embodiments, the present methods and compositionsaccelerate the process by which erythroid progenitors mature and arereleased into the circulation.

In some embodiments, the present methods and compositions increase therate of increase in hematocrit. In some embodiments, the present methodsand compositions maintain elevated hematocrits (e.g. of 25%, or 30%, or35%, or 40% or more) for a sustained period (e.g. about 1 month, orabout 2 months, or about 3 months, or about 4 months, or about 5 months,or about 6 months, or about 9 months).

In some embodiments, the present methods and compositions stimulate redblood cell production. In some embodiments, the present methods andcompositions stimulate division and differentiation of committederythroid progenitors in the bone marrow.

In some embodiments, the present chimeric EPO protein-relatedcompositions find use in decreasing the dose and/or frequency ofadministration when compared to wild type EPO and/or other EPO-basedagents. For instance, the present EPO-related compositions may find usein treatment regimens for the diseases disclosed herein (including,without limitation, one or more anemias) that involve administration ona monthly, or biweekly, or weekly basis. In some embodiments, therefore,the present EPO-related compositions reduce the need for daily, or, insome embodiments, weekly, administration. In some embodiments, thepresent EPO-related compositions require lower maintenance doses ascompared to wild type EPO or other EPO-based agents.

Certain embodiments of the present invention are particularly useful fortreating chemotherapy-induced anemia in cancer patients. In someembodiments, the present methods and compositions allows for continuedadministration of the chimeric protein after a cancer patient'schemotherapy is finished. In some embodiments, the present methods andcompositions allows for treatment of a cancer patient without dosereduction relative to a non-cancer patient. In some embodiments, thepresent methods and compositions allows for treatment of a cancerpatient receiving chemotherapy and considered curable. In variousembodiments, the cancer patient has one or more of a history of bloodclots, recent surgery, prolonged periods of bed rest or limitedactivity, and treatment with a chemotherapeutic agent.

In some embodiments, the present the present methods and compositionsprovide a fast and robust response that obviates the need for bloodtransfusion. For instance, in some embodiments, the present methods andcompositions allow for treatment of patients who do not consent to bloodtransfusions.

Kits

The invention also provides kits for the administration of any agentdescribed herein (e.g. the chimeric protein with or without variousadditional therapeutic agents). The kit is an assemblage of materials orcomponents, including at least one of the inventive pharmaceuticalcompositions described herein. Thus, in some embodiments, the kitcontains at least one of the pharmaceutical compositions describedherein.

The exact nature of the components configured in the kit depends on itsintended purpose. In one embodiment, the kit is configured for thepurpose of treating human subjects.

Instructions for use may be included in the kit. Instructions for usetypically include a tangible expression describing the technique to beemployed in using the components of the kit to effect a desired outcome,such as to treat anemia. Optionally, the kit also contains other usefulcomponents, such as, diluents, buffers, pharmaceutically acceptablecarriers, syringes, catheters, applicators, pipetting or measuringtools, bandaging materials or other useful paraphernalia as will bereadily recognized by those of skill in the art.

The materials and components assembled in the kit can be provided to thepractitioner stored in any convenience and suitable ways that preservetheir operability and utility. For example, the components can beprovided at room, refrigerated or frozen temperatures. The componentsare typically contained in suitable packaging materials. In variousembodiments, the packaging material is constructed by well-knownmethods, preferably to provide a sterile, contaminant-free environment.The packaging material may have an external label which indicates thecontents and/or purpose of the kit and/or its components.

Definitions

As used herein, “a,” “an,” or “the” can mean one or more than one.

Further, the term “about” when used in connection with a referencednumeric indication means the referenced numeric indication plus or minusup to 10% of that referenced numeric indication. For example, thelanguage “about 50” covers the range of 45 to 55.

An “effective amount,” when used in connection with medical uses is anamount that is effective for providing a measurable treatment,prevention, or reduction in the rate of pathogenesis of a disease ofinterest.

As used herein, something is “decreased” if a read-out of activityand/or effect is reduced by a significant amount, such as by at leastabout 10%, at least about 20%, at least about 30%, at least about 40%,at least about 50%, at least about 60%, at least about 70%, at leastabout 80%, at least about 90%, at least about 95%, at least about 97%,at least about 98%, or more, up to and including at least about 100%, inthe presence of an agent or stimulus relative to the absence of suchmodulation. As will be understood by one of ordinary skill in the art,in some embodiments, activity is decreased and some downstream read-outswill decrease but others can increase.

Conversely, activity is “increased” if a read-out of activity and/oreffect is increased by a significant amount, for example by at leastabout 10%, at least about 20%, at least about 30%, at least about 40%,at least about 50%, at least about 60%, at least about 70%, at leastabout 80%, at least about 90%, at least about 95%, at least about 97%,at least about 98%, or more, up to and including at least about 100% ormore, at least about 2-fold, at least about 3-fold, at least about4-fold, at least about 5-fold, at least about 6-fold, at least about7-fold, at least about 8-fold, at least about 9-fold, at least about10-fold, at least about 50-fold, at least about 100-fold, in thepresence of an agent or stimulus, relative to the absence of such agentor stimulus.

As referred to herein, all compositional percentages are by weight ofthe total composition, unless otherwise specified. As used herein, theword “include,” and its variants, is intended to be non-limiting, suchthat recitation of items in a list is not to the exclusion of other likeitems that may also be useful in the compositions and methods of thistechnology. Similarly, the terms “can” and “may” and their variants areintended to be non-limiting, such that recitation that an embodiment canor may comprise certain elements or features does not exclude otherembodiments of the present technology that do not contain those elementsor features.

Although the open-ended term “comprising,” as a synonym of terms such asincluding, containing, or having, is used herein to describe and claimthe invention, the present invention, or embodiments thereof, mayalternatively be described using alternative terms such as “consistingof” or “consisting essentially of.”

As used herein, the words “preferred” and “preferably” refer toembodiments of the technology that afford certain benefits, undercertain circumstances. However, other embodiments may also be preferred,under the same or other circumstances. Furthermore, the recitation ofone or more preferred embodiments does not imply that other embodimentsare not useful, and is not intended to exclude other embodiments fromthe scope of the technology.

The amount of compositions described herein needed for achieving atherapeutic effect may be determined empirically in accordance withconventional procedures for the particular purpose. Generally, foradministering therapeutic agents for therapeutic purposes, thetherapeutic agents are given at a pharmacologically effective dose. A“pharmacologically effective amount,” “pharmacologically effectivedose,” “therapeutically effective amount,” or “effective amount” refersto an amount sufficient to produce the desired physiological effect oramount capable of achieving the desired result, particularly fortreating the disorder or disease. An effective amount as used hereinwould include an amount sufficient to, for example, delay thedevelopment of a symptom of the disorder or disease, alter the course ofa symptom of the disorder or disease (e.g., slow the progression of asymptom of the disease), reduce or eliminate one or more symptoms ormanifestations of the disorder or disease, and reverse a symptom of adisorder or disease. Therapeutic benefit also includes halting orslowing the progression of the underlying disease or disorder,regardless of whether improvement is realized.

Effective amounts, toxicity, and therapeutic efficacy can be determinedby standard pharmaceutical procedures in cell cultures or experimentalanimals, e.g., for determining the LD50 (the dose lethal to about 50% ofthe population) and the ED50 (the dose therapeutically effective inabout 50% of the population). The dosage can vary depending upon thedosage form employed and the route of administration utilized. The doseratio between toxic and therapeutic effects is the therapeutic index andcan be expressed as the ratio LD50/ED50. In some embodiments,compositions and methods that exhibit large therapeutic indices arepreferred. A therapeutically effective dose can be estimated initiallyfrom in vitro assays, including, for example, cell culture assays. Also,a dose can be formulated in animal models to achieve a circulatingplasma concentration range that includes the 1050 as determined in cellculture, or in an appropriate animal model. Levels of the describedcompositions in plasma can be measured, for example, by high performanceliquid chromatography. The effects of any particular dosage can bemonitored by a suitable bioassay. The dosage can be determined by aphysician and adjusted, as necessary, to suit observed effects of thetreatment.

In certain embodiments, the effect will result in a quantifiable changeof at least about 10%, at least about 20%, at least about 30%, at leastabout 50%, at least about 70%, or at least about 90%. In someembodiments, the effect will result in a quantifiable change of about10%, about 20%, about 30%, about 50%, about 70%, or even about 90% ormore. Therapeutic benefit also includes halting or slowing theprogression of the underlying disease or disorder, regardless of whetherimprovement is realized.

As used herein, “methods of treatment” are equally applicable to use ofa composition for treating the diseases or disorders described hereinand/or compositions for use and/or uses in the manufacture of amedicaments for treating the diseases or disorders described herein.

EXAMPLES Example 1. Construction and Characterization of ChimericProteins Having Modified TNF

Chimeras were constructed which included mutant hTNF (R32W/S86T,Y87H/A145R, E146K, and Y87H/E146K mutations, for example, with referenceto SEQ ID NO: 5) fused N-terminally to either a Bcll10 (control) or ahCD20 Nanobody and a 20×GGS linker, and C-terminally to a 6×His-tag(referred to herein as “AcTakines”). Without wishing to be bound bytheory, it is believed that the mutant hTNF comprising the E146K singlemutation, Y87H/E146K double mutations, and/or R32W/S86T double mutationsselectively bound to hTNF-R1 and exhibited significantly reduced or nobinding to hTNF-R2. Without wishing to be bound by theory, it is alsobelieved that the mutant hTNF comprising the Y87H/A145R double mutationsselectively bound to hTNF-R2 and exhibited significantly reduced or nobinding to hTNF-R1. The chimeric AcTakines were cloned in the mammalianexpression vector pMet7 and expressed in HEK293T cells. The AcTakineconcentration in the cell supernatant was determined via hTNF ELISA (R&DSystems).

To investigate the bioactivity of the human TNF receptor-selectiveAcTakines via TNF-R1, a cytotoxicity assay involving MCF7 cells wasutilized (see panels A-B of FIGS. 1-3 ). MCF7 cells do not expressmembrane TNF-R2 (Dudich et al., 1999) and are highly sensitive toTNF-induced apoptosis. To allow targeting to MCF7 cells, cells werestably co-transfected with a plasmid containing the expression cassettefor human CD20 and a plasmid containing the neomycin resistance gene.Transfected cells were selected with G418 (1 mg/ml), followed by FACSsorting for hCD20-expressing cells. MCF7 and MCF7-hCD20 cells werestimulated for 72 hours with recombinant hTNF or AcTakines at theindicated doses, after which cell number was determined via luminescentdetection of ATP present per well (CellTiter-Glo, Promega).

Additionally, human TNF-R2 bioactivity was measured by using Raji Bcells (see panel C of FIGS. 1-3 ). Raji cells are derived from a Burkittlymphoma and express hCD20 and hTNF-R2, but not hTNF-R1 (Dudich et al.,1999). Kraus et al. (2003) demonstrated that stimulation of Raji cellswith TNF strongly induced the expression of p-opioid receptor (OPRM1),which could be inhibited by using a TNF-R2 blocking mAb. To determinethe bioactivity of receptor-selective AcTakines, Raji cells werestimulated for 24 hours at 1 ng/ml, whereafter cells were lysed and RNAwas isolated, and OPRM1 and GAPDH (as reference gene) mRNA expressionwere analyzed via qPCR. Ct-values were determined via the 2^(nd)derivative maximum method and calculation of relative OPRM1 versus GAPDHexpression was based on Vandesompele et al. (2002).

As shown in FIG. 1 , panels A and B, hTNF-R1 selective AcTakinescomprising the E146K or Y87H/E146K mutations displayed more than100-fold reduced bioactivity compared to wild-type recombinant hTNF onparental MCF7 cells, while targeting these AcTakines with hCD20Nanobodies to MCF7-hCD20 cells strongly increased bioactivity (48-foldfor E146K and 77-fold for Y87H/E146K, respectively). On Raji cells,however, these AcTakines displayed no bioactivity (see FIG. 1 , panelC).

Similarly, targeting of the hTNF-R1 selective AcTakine comprising theR32W/S86T mutations with a hCD20 Nanobody to MCF7-hCD20 cells increasedcytotoxicity by a factor of 16 compared to a Bcll10-targeted controlAcTakine, while targeting to Raji cells only induced a 2-fold increasein OPRM1 expression (see FIG. 2 , panels A-C).

As shown in FIG. 3 , panels A-C, the hTNF-R2 selective AcTakinecomprising the Y87H/A145R mutations had no bioactivity on MCF7 cells,not even by high affinity targeting to hCD20 to target over-expressingcells, while this AcTakine induced clear expression of OPRM1 in Rajicells. The Bcll10-targeted control AcTakine induced a 5-fold increase inOPRM1 expression compared to unstimulated cells, and expression wasfurther increased by targeting to hCD20.

In summary, inter alia, the hTNF AcTakines comprising one or more of theE146K, Y87H/E146K and R32W/S86T mutations displayed far superiorbioactivity and ‘reactivation by targeting’ in the hTNF-R1 bioassay thanthe hTNF-R2 bioassay. On the other hand, the hTNF AcTakine comprisingthe Y87H/A145R mutations displayed no bioactivity via hTNF-R1 while thisAcTakine had bioactivity via hTNF-R2 which could be further activated bytargeting in the hTNF-R2 bioassay.

Additional hTNF mutants were constructed (see Table below) and theirbinding to either hTNF-R1 or hTNF-R2 were characterized. Specifically,binding kinetics of the hTNF mutants were analyzed using an Octet RED96system (Pall Fortebio). Mutants were loaded onto HIS1K biosensors viatheir N-terminal 6×His tag and binding of soluble hTNF-R1 or -R2(Peprotech) at 8 different concentrations (½ dilution series startingfrom 500 nM) was measured. KD values were calculated using globalfitting with a 1:1 model (R²≥0.95). Results are presented below:

 mutant KD R1 (M) KD R2 (M) % WT R1 % WT R2 R1/R2 R2/R1 R32W S86T4.63E−08 — 22.11 — >> << R32W E146K 6.85E−08 — 14.96 — >> << Y87F1.16E−07 — 8.84 — > < E146K 5.79E−08 4.81E−06 17.71 0.49 35.95 0.03 L29SR32W 3.34E−08 1.26E−06 30.63 1.89 16.22 0.06 S86T 2.47E−08 2.20E−0741.53 8.32 4.99 0.20 L29S 4.53E−08 1.30E−07 22.60 18.25 1.24 0.81 Y87H6.51E−08 2.14E−07 15.73 11.09 1.42 0.71 Y87A — — — — — — D143N A145R —1.38E−07 — 17.22 << >> A145T E146D S147D 5.10E−06 4.78E−09 0.35 495.960.00 1413.08 A145R — 4.40E−08 — 53.93 << >> A145T S147D — 7.36E−09 —322.00 << >> WT 1.02E−08 2.37E−08

The binding activities of the hTNF mutants to hTNF-R1 were furthercharacterized using the cytotoxicity assay with MCF7 cells as describedpreviously (see panel A of FIG. 4 ). Additionally, the bindingactivities of the hTNF mutants to hTNF-R2 was further characterizedusing PC60-hTNFR2 cells (see panel B of FIG. 4 ). PC60-hTNFR2 cells arerat-mouse fusion hybridoma cells stably transfected with human TNF-R2sas described by Vandenabeele et al. (1995). Cells were cultured for 24hours with 2 ng/ml IL-1β and recombinant hTNF or His-TNF mutants atindicated doses, after which secreted rat GM-CSF in supernatant wasquantified via ELISA (R&D Systems).

As demonstrated in panels A and B of FIG. 4 , the hTNF-R1 selectivemutants L29 R32W, R32W S86T and E146K have similar or even increasedbioactivity compared to WT hTNF on MCF7 cells, while the R32W S86T andE146K mutants have no effect on rGM-CSF secretion by PC60-hTNFR2 cells.On the other hand, the hTNF-R2 selective mutant A145R has 370-folddecreased bioactivity on MCF7 cells but is able to induce secretion ofrGM-CSF by PC60-hTNFR2 cells at levels similar to WT hTNF.

Example 2. Construction and Characterization of Chimeric Proteins HavingModified EPO

Various chimeric proteins having modified EPO protein with attenuatedinteraction with EPOR were constructed. Specifically, the modified EPOincluded one or more of mutations selected from V11S, R14A, R14E, R14Q,Y151, K20E, T441, K451, K45D, V46A, F48G, K97A, K97E, S100E, S100T,R103A, R103E, R103H, R103N, R103Q, S104A, S1041, L105A, L108A, L108K,R110E, R143A, N147K, R150A, R150Q, R150E, G151A, L155A, and L155N, forexample, with reference to SEQ ID NO: 14.

Chimeric proteins having modified EPO protein with attenuatedinteraction with EphR were also constructed. For example, the modifiedEPO protein exhibited attenuated interaction with EphB4. Specifically,the modified EPO included one or more N-glycosylation and/or pegylationmodifications at amino acids R14, K97, R103, Q115, 1119, E123, A128,R131, E55, E72, R76, K83, and S85. Additionally, chimeric proteins withmodified EPO protein having mutations in the CD-loop were alsoconstructed. Such modified EPO proteins included the sequenceGGPPGSGKGSPGG (SEQ ID NO: 256) at amino acid positions 119-128 or thesequence GGSGGSGG (SEQ ID NO: 258) at amino acid positions 113-120.Further, chimeric proteins with modified EPO protein having mutations inhelix B were also constructed. Specifically, the modified EPO proteinincluded one or more mutations at amino acids Q58, E62, Q65, L69, E72,R76, A79, and L80.

The various chimeric proteins having modified EPO protein aretransfected into cells and their activities are characterized.

EQUIVALENTS

While the invention has been described in connection with specificembodiments thereof, it will be understood that it is capable of furthermodifications and this application is intended to cover any variations,uses, or adaptations of the invention following, in general, theprinciples of the invention and including such departures from thepresent disclosure as come within known or customary practice within theart to which the invention pertains and as may be applied to theessential features hereinbefore set forth and as follows in the scope ofthe appended claims.

Those skilled in the art will recognize, or be able to ascertain, usingno more than routine experimentation, numerous equivalents to thespecific embodiments described specifically herein. Such equivalents areintended to be encompassed in the scope of the following claims.

INCORPORATION BY REFERENCE

All patents and publications referenced herein are hereby incorporatedby reference in their entireties.

The publications discussed herein are provided solely for theirdisclosure prior to the filing date of the present application. Nothingherein is to be construed as an admission that the present invention isnot entitled to antedate such publication by virtue of prior invention.

As used herein, all headings are simply for organization and are notintended to limit the disclosure in any manner. The content of anyindividual section may be equally applicable to all sections.

REFERENCES

-   Dudich E., Semenkova L., Dudich I., Gorbatova E., Tochtamisheva N.,    Tatulov N., Nikolaeva M. and Sukhikh G. alpha-fetoprotein causes    apoptosis in tumor cells via a pathway independent of CD95, TNFR1    and TNFR2 through activation of caspase-3-like proteases. Eur. J.    Biochem. 266, 750-61 (1999)-   Kraus J., Börner C., Giannini E. and Höllt V. The Role of Nuclear    Factor kB in Tumor Necrosis Factor-Regulated Transcription of the    Human p-Opioid Receptor Gene. Mol. Pharmacol. 64, 876-884 (2003)-   Vandesompele J., De Preter K., Pattyn F., Poppe B., Van Roy N., De    Paepe A. and Speleman F. Accurate normalization of real-time    quantitative RT-PCR data by geometric averaging of multiple internal    control genes. Genome Biol. 3 (2002)-   Vandenabeele P., Declercq W., Vanhaesebroeck B., Grooten J. and    Fiers, W. (1995b). Both TNF receptors are required for TNF-mediated    induction of apoptosis in PC60 cells. J. Immunol. 154, 2904-2913    (1995).

What is claimed is:
 1. A chimeric protein, comprising: (a) a modifiedhuman TNF-α having a reduced affinity for TNF-R1 and TNF-R2 as comparedto wild type human TNF-α, and (b) a targeting moiety comprising arecombinant heavy-chain-only antibody (VHH) or a single-chain antibody(scFv) directed against CD20, wherein the modified human TNF-α has atleast 95% identity to the amino acid sequence of SEQ ID NO: 5 andcomprises the mutations (i) R32W and S86T or (ii) L29S and R32W, andwherein the mutations to the human TNF-α confer: (a) reduced, butrestorable, affinity for TNF-R1; and (b) reduced, but not restorable,affinity for TNF-R2, wherein the targeting moiety directed against CD20selectively restores the affinity of the modified human TNF-α for TNF-R1but not TNF-R2.
 2. The chimeric protein of claim 1, further comprising alinker, wherein the linker sequence comprises GGS or GGS repeats.
 3. Thechimeric protein of claim 1, wherein the modified human TNF-α has atleast 97% identity to the amino acid sequence of SEQ ID NO:
 5. 4. Thechimeric protein of claim 1, wherein the modified human TNF-α is atrimeric single polypeptide chain construct.
 5. The chimeric protein ofclaim 1, wherein an agonistic activity or an antagonist activity of themodified human TNF-α is attenuated at TNF-R1.
 6. A chimeric protein,comprising: (a) a modified human TNF-α having a reduced affinity forTNF-R1 and TNF-R2 as compared to wild type human TNF-α and (b) atargeting moiety comprising a VHH or a scFv directed against CD20,wherein the modified human TNF-α has at least 95% identity to the aminoacid sequence of SEQ ID NO: 5 and comprises the following mutations: i.R32W and 586T; ii. R32W and E146K; or iii. L29S and R32W; wherein thetargeting moiety directed against CD20 selectively restores the affinityof the modified human TNF-α for TNF-R1 but not TNF-R2.
 7. The chimericprotein of claim 6, further comprising a linker, wherein the linkersequence comprises GGS or GGS repeats.
 8. The chimeric protein of claim6, wherein the modified human TNF-α has at least 97% identity to theamino acid sequence of SEQ ID NO:
 5. 9. The chimeric protein of claim 6,wherein the modified human TNF-α is a trimeric single polypeptide chainconstruct.
 10. The chimeric protein of claim 6, wherein an agonisticactivity or an antagonist activity of the modified human TNF-α isattenuated at TNF-R1.